Enzyme Triggered Redox Altering Chemical Elimination (E-Trace) Immunoassay

ABSTRACT

Described are methods for detecting a target analyte in a test sample by providing a solid support comprising an electrode comprising: (i) a self-assembled monolayer (SAM), (ii) a covalently attached electroactive active moiety (EAM) comprising a transition metal complex comprising a self-immolative moiety and a peroxide sensitive moiety (PSM), wherein said EAM has a first E 0 ; then contacting the target analyte and said solid support under conditions wherein said target analyte reacts with a peroxide generating enzyme to generate peroxide and said self-immolative moiety is removed such that said EAM has a second E 0  test sample; and then detecting said second E 0  as an indication of the presence of said target analyte. Also provided are compositions for use in the preceding methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of, and priority to, U.S.Provisional Patent Application No. 61/434,122, filed Jan. 19, 2011, andU.S. Provisional Patent Application No. 61/523,679, filed Aug. 15, 2011,the entire disclosures of which are hereby incorporated by reference intheir entirety.

FIELD OF THE INVENTION

The invention relates to novel compositions and methods for thedetection of target analytes using change in E⁰ of a transitional metalcomplex.

BACKGROUND OF THE INVENTION

Electron transfer reactions are crucial steps in a wide variety ofbiological transformations ranging from photosynthesis or aerobicrespiration. Studies of electron transfer reactions in both chemical andbiological systems have led to the development of a large body ofknowledge and a strong theoretical base, which describes the rate ofelectron transfer in terms of a small number of parameters.

Electronic tunneling in proteins and other biological molecules occursin reactions where the electronic interaction of the redox centers isrelatively weak. Semiclassical theory reaction predicts that thereaction rate for electron transfer depends on the driving force (−ΔG°),a nuclear reorganization parameter (A), and the electronic-couplingstrength between the reactants and products at the transition state(H_(AB)), according to the following equation:

k _(ET)=(4π³ /h ² λk _(B) T)^(1/2)(H _(AB))²exp[(−ΔG°+λ)2/λk _(B) T]

The nuclear reorganization energy, λ, in the equation above is definedas the energy of the reactants at the equilibrium nuclear configurationof the products. For electron transfer reactions in polar solvents, thedominant contribution to λ arises from the reorientation of solventmolecules in response to the change in charge distribution of thereactants. The second component of λ comes from the changes in bondlengths and angles due to changes in the oxidation state of the donorsand acceptors.

Previous work describes using change in reorganization energy, λ, as thebasis of novel sensor. See for example, U.S. Pat. Nos. 6,013,459,6,013,170, 6,248,229, and 7,267,939, all herein incorporated byreference in their entirety. The methods generally comprise binding ananalyte to or near a redox active complex. The redox active complexcomprises at least one redox active molecule and a capture ligand whichwill bind the target analyte, and the complex is bound to an electrode.Upon analyte binding, the reorganization energy of the redox activemolecule changes, e.g. the E⁰ is altered, such that measuring the changein E⁰ allows the detection of the target analyte.

It is an object of the present invention to provide composition andmethods for the detection of target analytes using alteration in thesolvent reorganization energy, corresponding to changes in the E⁰ ofredox active molecules.

SUMMARY OF THE INVENTION

The present invention to provide composition and methods for thedetection of target analytes using changes in standard potentials of theE⁰ of redox active molecules upon peroxide-triggered change in theapparent formal potential.

In one aspect, the invention provides compositions and methods for thedetection of target analytes in a test sample. Thus, the inventionprovides a solid support comprising an electrode comprising: aself-assembled monolayer (SAM). (ii) a covalently attached electroactiveactive moiety (EAM) comprising a transition metal complex comprising aself-immolative moiety (SIM) and a peroxide sensitive moiety (PSM),wherein said EAM has a first E⁰ and a capture binding ligand that bindsthe analyte, and a self-assembled monolayer (SAM).

The methods proceed by contacting the target analyte and the solidsupport, under conditions wherein the target analyte binds the capturebinding ligand to form a first complex, and contacting the first complexwith a soluble capture ligand that binds the target analyte, wherein thesoluble capture ligand comprises a peroxide generating moiety to form asecond complex. A peroxide substrate is added to the second complexunder conditions that peroxide is generated and the self-immolativemoiety is removed such that the EAM has a second E⁰. The second E⁰ isthen detected as an indication of the presence of said target.

In another aspect, the invention provides methods for detecting a targetanalyte in a test sample, said method comprising: (a) providing a solidsupport comprising an electrode comprising: (i) a self-assembledmonolayer (SAM); (ii) a covalently attached electroactive active moiety(EAM) comprising a transition metal complex comprising a self-immolativemoiety and a peroxide sensitive moiety (PSM), wherein said EAM has afirst E⁰; (iii) a peroxide-generating enzyme that generates peroxide inthe presence of a substrate for said peroxide-generating enzyme; (b)contacting said target analyte and said solid support in the presence ofan enzyme and an additional substrate for said enzyme, under conditionswherein if said analyte is present, the substrate for saidperoxide-generating enzyme is formed, said peroxide is generated andsaid self-immolative moiety is removed such that said EAM has a secondE⁰; and (c) detecting said second E⁰ as an indication of the presence ofsaid target analyte.

In another aspect, the invention provides methods for detecting a targetanalyte in a test sample, said method comprising: (a) providing a solidsupport comprising an electrode comprising: (i) a self-assembledmonolayer (SAM); (ii) a covalently attached electroactive active moiety(EAM) comprising a transition metal complex comprising a self-immolativemoiety and a peroxide sensitive moiety (PSM), wherein said EAM has afirst E⁰, (iii) a peroxide generating enzyme that generates peroxide inthe presence of said target analyte; (b) contacting said target analyteand said solid support, under conditions wherein said target analytereacts with said enzyme to generate peroxide and said self-immolativemoiety is removed such that said EAM has a second E⁰; and (c) detectingsaid second E⁰ as an indication of the presence of said target analyte.

In another aspect, the invention provides methods for detecting a targetanalyte in a test sample, said method comprising: (a) providing a solidsupport comprising an electrode comprising: (i) a self-assembledmonolayer (SAM); (ii) a covalently attached electroactive active moiety(EAM) comprising a transition metal complex comprising a self-immolativemoiety and a peroxide sensitive moiety (PSM), wherein said EAM has afirst E⁰, (b) contacting said target analyte and said solid support inthe presence of an enzyme and optionally an additional substrate forsaid enzyme, under conditions wherein if said analyte is present a firstcomplex is formed; (c) contacting said first complex with a peroxidegenerating enzyme under conditions wherein if said first complex isformed, said enzyme is attached to said electrode; (d) providing asubstrate for said enzyme such that peroxide is generated and saidself-immolative moiety is removed such that said EAM has a second E⁰;and (e) detecting said second E⁰ as an indication of the presence ofsaid target analyte.

In another aspect, the invention provides methods for detecting a targetanalyte in a test sample, said method comprising: (a) providing a solidsupport comprising an electrode comprising: (i) a self-assembledmonolayer (SAM); (ii) a covalently attached electroactive active moiety(EAM) comprising a transition metal complex comprising a self-immolativemoiety and a peroxide sensitive moiety (PSM), wherein said EAM has afirst E⁰; (b) contacting said target analyte and said solid support inthe presence of an enzyme and an additional substrate for said enzyme,under conditions wherein if said analyte is present, a substrate for aperoxide-generating enzyme is formed; (c) contacting said substrate fora peroxide-generating enzyme with a peroxide generating enzyme underconditions wherein if said substrate for a peroxide-generating enzyme isformed, peroxide is generated and said self-immolative moiety is removedsuch that said EAM has a second E⁰; and (d) detecting said second E⁰ asan indication of the presence of said target analyte.

In another aspect, the invention provides composition comprising a solidsupport comprising: (a) an electrode comprising: (i) a self-assembledmonolayer (SAM); (ii) a covalently attached electroactive active moiety(EAM) comprising a transition metal complex comprising a self-immolativemoiety and a peroxide sensitive moiety (PSM), wherein said EAM has afirst E⁰ when said self-immolative moiety is covalently attached to saidEAM and a second E⁰ when said self-immolative moiety is absent; (iii) aperoxide-generating enzyme that generates peroxide in the presence of asubstrate for said peroxide-generating enzyme; and (b) a soluble enzymethat generates said substrate for said peroxide-generating enzyme in thepresence of a target analyte and an additional substrate or said solubleenzyme.

In another aspect, the invention provides composition comprising a solidsupport comprising: (a) an electrode comprising: (i) a self-assembledmonolayer (SAM); (ii) a covalently attached electroactive active moiety(EAM) comprising a transition metal complex comprising a self-immolativemoiety and a peroxide sensitive moiety (PSM), wherein said EAM has afirst E⁰ when said self-immolative moiety is covalently attached to saidEAM and a second E⁰ when said self-immolative moiety is absent; (b) asoluble peroxide-generating enzyme that generates peroxide in thepresence of a substrate for said peroxide-generating enzyme; (c) asoluble enzyme that generates said substrate for saidperoxide-generating enzyme in the presence of a target analyte and anadditional substrate of said soluble enzyme.

In another aspect, the invention provides methods for detecting ATP in atest sample, said method comprising: (a) providing a solid supportcomprising an electrode comprising: (i) a self-assembled monolayer(SAM), (ii) a covalently attached electroactive active moiety (EAM)comprising a transition metal complex comprising a self-immolativemoiety and a peroxide sensitive moiety (PSM), wherein said EAM has afirst E⁰, and (iii) a capture binding ligand that is a substrate of anintermediary enzyme; (b) contacting said test sample and said solidsupport in the presence of said intermediary enzyme and a first memberof a specific binding pair, under conditions wherein if said targetanalyte is present in said test sample, said intermediary enzymephosphorylates and conjugates said first member of the specific bindingpair to said capture binding ligand to form a first complex; (c)contacting said first complex with a soluble capture ligand that bindssaid first complex to form a second complex, wherein said solublecapture ligand comprises a peroxide generating moiety; (d) adding aperoxide substrate to said second complex under conditions that peroxideis generated and said self-immolative moiety is removed such that saidEAM has a second E⁰; and (e) detecting said second E⁰ as an indicationof the presence of said ATP.

In one embodiment of the preceding aspect, prior to said step (c), awashing step is performed.

In an embodiment of the preceding aspect and any embodiment thereof,prior to step (d), a washing step is performed.

In an embodiment of the preceding aspect and any embodiment thereof,said intermediary enzyme and said first member of said specific bindingis physisorbed onto the onto said SAM. “Physisorbed” as used hereinmeans the enzyme is adsorbed onto the referenced surface (e.g.,electrode surface or SAM surface) via non-covalent interactions, such ashydrophobic or ionic interactions.

In an embodiment of the preceding aspect and any embodiment thereof,said solid support comprises an array of electrodes.

In an embodiment of the preceding aspect and any embodiment thereof,said transition metal is selected from the group consisting of iron,ruthenium and osmium.

In an embodiment of the preceding aspect and any embodiment thereof,said EAM is a ferrocene.

In another aspect, the invention provides methods for detecting ATP in atest sample, said method comprising: (a) providing a solid supportcomprising an electrode comprising: (i) a self-assembled monolayer(SAM); (ii) a covalently attached electroactive active moiety (EAM)comprising a transition metal complex comprising a self-immolativemoiety and a peroxide sensitive moiety (PSM), wherein said EAM has afirst E⁰, (b) contacting said test sample and said solid support in thepresence of an intermediary enzyme and a substrate of said intermediaryenzyme, under conditions wherein if said ATP is present in said testsample, said intermediary enzyme phosphorylates said substrate of saidintermediary enzyme to form a peroxide substrate; (c) contacting saidperoxide substrate with said peroxide-generating enzyme under conditionsthat peroxide is generated and said self-immolative moiety is removedsuch that said EAM has a second E⁰; and (d) detecting said second E⁰ asan indication of the presence of said ATP.

In an embodiment of the preceding aspect and any embodiment thereof,prior steps (b) and (c) are carried out simultaneously.

In an embodiment of the preceding aspect and any embodiment thereof,prior wherein said intermediary enzyme is glycerol kinase and saidsubstrate of said intermediary enzyme is glycerol.

In an embodiment of the preceding aspect and any embodiment thereof,prior wherein said peroxide generating moiety is a glycerol-3-phosphateoxidase.

In an embodiment of the preceding aspect and any embodiment thereof,said solid support comprises an array of electrodes.

In an embodiment of the preceding aspect and any embodiment thereof,said transition metal is selected from the group consisting of iron,ruthenium and osmium.

In an embodiment of the preceding aspect and any embodiment thereof,said EAM is a ferrocene.

In another aspect, the invention provides methods for detecting ATP in atest sample, said method comprising: (a) providing a solid supportcomprising an electrode comprising: (i) a self-assembled monolayer(SAM); (ii) a covalently attached electroactive active moiety (EAM)comprising a transition metal complex comprising a self-immolativemoiety and a peroxide sensitive moiety (PSM), wherein said EAM has afirst E⁰; and (iii) a peroxide-generating enzyme; (b) contacting saidtest sample and said solid support in the presence of an intermediaryenzyme and a substrate of said intermediary enzyme, wherein said ATP isa co-factor of said intermediary enzyme, under conditions wherein ifsaid ATP is present in said test sample, said intermediary enzymephosphorylates said substrate of said intermediary enzyme to form asubstrate of said peroxide-generating enzyme; (c) contacting saidsubstrate of said peroxide-generating enzyme with saidperoxide-generating enzyme under conditions that peroxide is generatedand said self-immolative moiety is removed such that said EAM has asecond E⁰; and (d) detecting said second E⁰ as an indication of thepresence of said ATP.

In an embodiment of the preceding aspects, prior steps (b) and (c) arecarried out separately.

In an embodiment of the preceding aspect and any embodiment thereof,prior steps (b) and (c) are carried out simultaneously.

In an embodiment of the preceding aspect and any embodiment thereof,prior wherein said intermediary enzyme is glycerol kinase and saidsubstrate of said intermediary enzyme is glycerol.

In an embodiment of the preceding aspect and any embodiment thereof,prior wherein said peroxide generating moiety is a glycerol-3-phosphateoxidase.

In an embodiment of the preceding aspect and any embodiment thereof,said solid support comprises an array of electrodes.

In an embodiment of the preceding aspect and any embodiment thereof,said transition metal is selected from the group consisting of iron,ruthenium and osmium.

In an embodiment of the preceding aspect and any embodiment thereof,said EAM is a ferrocene.

In another aspect, the invention provides compositions comprising asolid support comprising: (a) an electrode comprising: (i) aself-assembled monolayer (SAM); (ii) a covalently attached electroactiveactive moiety (EAM) comprising a transition metal complex comprising aself-immolative moiety and a peroxide sensitive moiety (PSM), whereinsaid EAM has a first E⁰ when said self-immolative moiety is covalentlyattached to said EAM and a second E⁰ when said self-immolative moiety isabsent; (iii) a capture binding ligand comprising a substrate of anATP-dependent intermediary enzyme; and (d) a soluble capture ligandcomprising a peroxide generating moiety.

In another aspect, the invention provides compositions comprising asolid support comprising: (a) an electrode comprising: (i) aself-assembled monolayer (SAM); (ii) a covalently attached electroactiveactive moiety (EAM) comprising a transition metal complex comprising aself-immolative moiety and a peroxide sensitive moiety (PSM), whereinsaid EAM has a first E⁰ when said self-immolative moiety is covalentlyattached to said EAM and a second E⁰ when said self-immolative moiety isabsent; and (iii) a peroxide generating enzyme.

In another aspect, the invention provides methods for detecting a targetanalyte in a test sample, said method comprising: (a) providing a solidsupport comprising an electrode comprising: (i) a self-assembledmonolayer (SAM), (ii) a covalently attached electroactive active moiety(EAM) comprising a transition metal complex comprising a self-immolativemoiety and a peroxide sensitive moiety (PSM), wherein said EAM has afirst E⁰, and (b) contacting said target analyte and said solid supportunder conditions wherein said target analyte reacts with a peroxidegenerating enzyme to generate peroxide and said self-immolative moietyis removed such that said EAM has a second E⁰ test sample; and (c)detecting said second E⁰ as an indication of the presence of said targetanalyte.

In another aspect, the invention provides methods for detecting a targetanalyte in a test sample, said method comprising: (a) providing a solidsupport comprising an electrode comprising: (i) a self-assembledmonolayer (SAM), (ii) a covalently attached electroactive active moiety(EAM) comprising a transition metal complex comprising a self-immolativemoiety and a peroxide sensitive moiety (PSM), wherein said EAM has afirst E⁰, and (b) contacting said target analyte and said solid supportin the presence of an intermediary enzyme and optionally an additionalsubstrate for said enzyme, under conditions wherein if said analyte ispresent, the target analyte and said optional additional substratereacts with the intermediary enzyme to form a first complex; (c)contacting said first complex with said peroxide generating enzyme underconditions wherein if said first complex is formed, said first complexreacts with said peroxide generating enzyme to generate peroxide andsaid self-immolative moiety is removed such that said EAM has a secondE⁰ test sample; and (d) detecting said second E⁰ as an indication of thepresence of said target analyte.

In an embodiment of either of the two previous aspects, said peroxidegenerating enzyme is immobilized or physisorbed onto the solid support,the electrode, or SAM.

In an embodiment of either of the two previous aspects, and anyembodiment thereof, said intermediary enzyme is immobilized orphysisorbed onto the solid support, the electrode, or SAM.

In an embodiment of either of the two previous aspects, and anyembodiment thereof, wherein steps (b) and (c) are carried outseparately.

In an embodiment of either of the two previous aspects, and anyembodiment thereof, steps (b) and (c) are carried out simultaneously.

In an embodiment of either of the two previous aspects, and anyembodiment thereof, said target analyte is ATP, said intermediary enzymeis glycerol kinase and said substrate of said intermediary enzyme isglycerol, and said peroxide generating moiety is glycerol-3-oxidase.

In an embodiment of either of the two previous aspects, and anyembodiment thereof, said target analyte is NADH and said peroxidegenerating moiety is NADH oxidase (NAOX).

In an embodiment of either of the two previous aspects, and anyembodiment thereof, said solid support comprises an array of electrodes.

In an embodiment of either of the two previous aspects, and anyembodiment thereof, said transition metal is selected from the groupconsisting of iron, ruthenium and osmium.

In an embodiment of either of the two previous aspects, and anyembodiment thereof, said EAM is a ferrocene.

In an embodiment of either of the two previous aspects, and anyembodiment thereof, said detecting is done amperommetrically at eachfirst and second E⁰.

In another aspect, the invention provides compositions comprising asolid support comprising: (a) an electrode comprising: (i) aself-assembled monolayer (SAM); (ii) a covalently attached electroactiveactive moiety (EAM) comprising a transition metal complex comprising aself-immolative moiety and a peroxide sensitive moiety (PSM), whereinsaid EAM has a first E⁰ when said self-immolative moiety is covalentlyattached to said EAM and a second E⁰ when said self-immolative moiety isabsent; (iii) a capture binding ligand comprising a substrate of anATP-dependent intermediary enzyme; and (d) a soluble capture ligandcomprising a peroxide generating moiety.

In another aspect, the invention provides compositions comprising asolid support comprising: (a) an electrode comprising: (i) aself-assembled monolayer (SAM); (ii) a covalently attached electroactiveactive moiety (EAM) comprising a transition metal complex comprising aself-immolative moiety and a peroxide sensitive moiety (PSM), whereinsaid EAM has a first E⁰ when said self-immolative moiety is covalentlyattached to said EAM and a second E⁰ when said self-immolative moiety isabsent; and (iii) a peroxide generating enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Structure of electroactive molecule (EAM) 1 and mechanism ofperoxide-induced ligand dissociation. The change in ligand electronicsis responsible for the shift in redox potential.

FIG. 2. The synthetic scheme of one of the embodiments of the inventionas depicted in FIG. 1.

FIG. 3. Solution CV data for EAM 1 and a control compound following H₂O₂induced cleavage of the POM ligand. The change in E⁰ following theself-immolative process is 331 mV. Experiments were run in THF withTBAClO₄ (150 mM) supporting electrolyte using a carbon workingelectrode, Ag/AgCl reference electrode, and a Pt wire counter electrode.

FIG. 4. Overlaid cyclic voltammograms from a SAM of EAM 1 before(dotted) and after solid incubation with 1 mM hydrogen peroxide inNaHCO₃ buffer (pH 8.5) for 10 min, followed by a 5-min wash in Na₂CO₃buffer (pH 10.1; lower peaks). Supporting electrolyte was 1M LiClO₄,silver quasi reference electrode, platinum wire counter electrode. Scanrate was 10000 mV/s.

FIG. 5. Overlaid cyclic voltammograms from a SAM of EAM 1 before(dotted) and after (solid) incubation with 1 mM glucose and 100 uMglucose oxidase in NaHCO₃ buffer (pH 8.5) for 10 min, followed by a 5min wash in Na₂CO₃ buffer (pH 10.1). Supporting electrolyte was 1MLiClO₄, silver quasi reference electrode, platinum wire counterelectrode. Scan rate was 10000 mV/s.

FIG. 6. Sample self-immolative spacer groups based on substitutedquinone methides.

FIG. 7. Cyclic voltamogram for SAM of EAM 1 following antibody sandwichformation with human cardiac troponin I (10 ng/mL) before (dotted) andafter (solid) incubation with glucose for 10 min. Inset shows the peakat −0.10V magnified.

FIG. 8. Depicts a variety of self-immolative moieties which find use inthe present invention. “PSM” stands for “peroxide sensitive moiety” and“EAM” stands for “electroactive moiety”. As is shown in the figures, avariety of monomeric self-immolative moieties (sometimes referred toherein as “SIM”) can be used; FIG. 8A depicts a first type ofself-immolative moiety, which relies on the PSM contributing an —OHgroup upon contact with peroxide, resulting a phenol-based linker thatreleases from the EAM. n can be an integer of 1 or higher, with from 1to 5 finding particular use in the invention. m is an integer of atleast one; as will be appreciated by those in the art, m will depend onthe transitional metal complex used and the number of positions in theEAM; for example, when a metallocene such as ferrocene is used, therecan be up to 5 PSM-SIMs per cyclopentadienyl ring, with at least one ofthe positions on one of the rings being used for attachment to theelectrode. FIGS. 8B, 8C and 8D show multimers of SIMs. X can be —NH— or—O—.

FIG. 9. Depicts additional peroxide sensitive moieties. Figure A depictsthe PSB ether (parasiletanylbenzyl) moiety and Figure B depicts thepentafluorophenylsulfonate (PFPS) moiety. As shown in Figure (next),there can be more than one self-immolative moiety per EAM and/or morethan one PSM-SIM per EAM. As for the boron containing PSMs, there can bemultiple PSB ethers or PFPS moieties per EAM as well.

FIG. 10. Depicts a ferrocene that has R groups. The moiety shown has theattachment linker and the self-immolative moiety and the peroxidesensitive moiety on different rings, although as described herein, theycan be on the same ring. In addition, any or all of the R groups can bean additional -SIM-PSM substituent, as well as traditional substituents(alkyl (including substituted alkyl, heteroalkyl, cyclic alkyl, etc.),aryl (including substituted aryl and heteroaryl), etc.).

FIG. 11 is similar to FIG. 1 in showing the reaction mechanism for arepresentative ferrocene based EAM that undergoes a peroxide-triggeredchange in the apparent formal potential. (A) is the starting ferrocenylEAM that contains an electron-withdrawing carbamate-linked boronateester-substituted ligand. (B) Shows that the reaction with peroxideleads to an electron-donating amino ligand on the ferrocene whichchanges the redox potential (E⁰).

FIG. 12 depicts a representative metabolite assay for the detection ofNADH in a sample. In this particular embodiment, the NADH oxidase(nicotinamide adenine dinucleotide phosphate-oxidase) is attached to theelectrode using a biotin-streptavidin system, although as will beappreciated by those in the art, any number of additional methods can beused, including the use of the NHS or maleimide system outlined inPCT/US2008/080379, hereby incorporated by reference in its entirety. Inthis embodiment, the NADH oxidase is attached via an amine or otherfunctional group, such as the N-terminus, using activation chemistry.

FIG. 13 depicts one embodiment of the electrochemical detection of ATPas a metabolite in a sample. In this embodiment, the sensor is based ona SAM-modified gold electrode that contains peroxide-triggeredelectroactive molecules (EAMs, sometimes alternatively referred toherein as “redox active moieties” (RAMs)) and acceptor peptide (AP)substrates for BirA (biotin ligase, in this case from E. coli) withphysisorbed biotin and BirA (FIG. 13A). FIG. 13B depicts that BirAcatalyzes the biotinlyation of the A. After washing, a glucoseoxidase-streptavidin conjugate is further conjugated to the biotinattached to the AP. FIG. 13C depicts the reaction when glucose is addedand the peroxide generated reacts with the EAMs with the self-immolativemoiety, resulting in a change in the E⁰.

FIG. 14. Schematic representation of an electrochemical ATP assay. G3POmay be in solution or immobilized at the SAM interface viabiotin-streptavidin binding or by adsorption to the SAM interface.

FIG. 15. Electrochemical data for NADH detection as described in Example4.

FIG. 16. Electrochemical data for ATP detection as described in Example5.

DETAILED DESCRIPTION OF THE INVENTION

U.S. patent application Ser. Nos. 12/253,828, 12/253,875, 12/853,204,61/332,565, 61/347,121, 61/394,458, 61/366,013 and 61/407,828 are allincorporated by reference in their entirety.

Overview

The present invention is directed to electronic methods of detectingtarget analytes such that upon binding of the target analyte a shift inelectrochemical potential is seen. This mechanism has been generallydescribed in U.S. Pat. Nos. 7,595,153, 7,759,073 and 7,713,711, and U.S.patent application Ser. No. 12/253,828, filed Oct. 17, 2008; U.S. patentapplication Ser. No. 12/253,875, filed Oct. 17, 2008; U.S. ProvisionalPatent Application No. 61/332,565, filed May 7, 2010; U.S. ProvisionalPatent Application No. 61/347,121, filed May 21, 2010; and U.S.Provisional Patent Application No. 61/366,013, filed Jul. 20, 2010, allof which are expressly incorporated by reference in their entirety.

The assay relies on the use of an electroactive moiety (“EAM”), which isattached to the electrode and comprises a self-immolative moiety, whosepresence gives the EAM a first E⁰, whose absence, upon irreversiblecleavage, gives the EAM a second E⁰. The electrode also contains capturebinding ligands that will bind the target analyte upon its introduction.A soluble capture ligand is introduced, which also binds the targetanalyte and comprises a peroxide generating moiety, such as a glucoseoxidase enzyme. Upon the addition of oxygen and a substrate for theperoxidase generating moiety (e.g. an oxygen saturated buffer andglucose, in the case of a glucose oxidase enzyme as the peroxidasegenerating moiety) peroxide is generated, attacking the self-immolativemoiety and causing the removal of the self-immolative moiety from theEAM, which results in a change in the E⁰ of the EAM. This difference isdetected, and if such a change occurs, it is an indication of thepresence of the target analyte.

Thus, to determine whether a target analyte is present in the sample,the sample is applied to the detection electrode surface, optionallywashed, and an oxidase enzyme-conjugated secondary binding ligand (forexample, an antibody) that binds an alternative epitope of the targetanalyte is added, creating a “sandwich assay” format with the target.The surface is optionally washed, and treated with an oxygen-saturatedbuffer containing a high concentration of glucose. The presence of thesubstrate oxidase enzyme (sometimes referred to herein as “SOX”) on thesurface results in the enzymatic creation of hydrogen peroxide insolution which diffuses to the monolayer surface and triggers a chemicalelimination reaction (“self-immolative” reaction) in the immobilizedEAMs. This irreversible elimination reaction changes the electronicenvironment of the EAM, for example by altering the “R” groups (e.g.substituent groups) of the transition metal complex, thus shifting theapparent formal potential of the EAM to a second E⁰ to signal thepresence of the target.

In other aspects, the target analyte may be a substrate for anintermediary enzyme and an optional additional substrate whichultimately generates a substrate for a peroxide generating moiety.Examples of such substrates include, but are not limited to, ATP andNADH. For example, ATP is a substrates for an intermediary enzyme suchas, but not limited to, biotin ligase (BirA) and glycerol kinase (GK)which utilize ATP as a substrate to phosphorylate their respectiveacceptor protein and glycerol substrates. An “intermediary enzyme” asused herein, refers to an enzyme that utilizes a target analytes as asubstrate.

Accordingly, the present invention provides methods and compositions fordetecting target analytes in samples.

Target Analytes

By “target analyte” or “analyte” or grammatical equivalents herein ismeant any molecule, compound or particle to be detected. Target analytesbind to binding ligands (both capture and soluble binding ligands), asis more fully described below. As will be appreciated by those in theart, a large number of analytes may be detected using the presentmethods; basically, any target analyte for which a binding ligand,described below, may be made may be detected using the methods of theinvention.

Suitable analytes include organic and inorganic molecules, includingbiomolecules. In a preferred embodiment, the analyte may be anenvironmental pollutant (including pesticides, insecticides, toxins,etc.); a chemical (including solvents, polymers, organic materials,etc.); therapeutic molecules (including therapeutic and abused drugs,antibiotics, etc.); biomolecules (including hormones, cytokines,proteins, lipids, carbohydrates, cellular membrane antigens andreceptors (neural, hormonal, nutrient, and cell surface receptors) ortheir ligands, etc); whole cells (including procaryotic (such aspathogenic bacteria) and eukaryotic cells, including mammalian tumorcells); viruses (including retroviruses, herpesviruses, adenoviruses,lentiviruses, etc.); and spores; etc.

In some embodiments, the target analyte is a protein. As will beappreciated by those in the art, there are a large number of possibleproteinaceous target analytes that may be detected using the presentinvention. By “proteins” or grammatical equivalents herein is meantproteins, oligopeptides and peptides, derivatives and analogs, includingproteins containing non-naturally occurring amino acids and amino acidanalogs, and peptidomimetic structures. The side chains may be in eitherthe (R) or the (S) configuration. In a preferred embodiment, the aminoacids are in the (S) or L configuration. As discussed below, when theprotein is used as a binding ligand, it may be desirable to utilizeprotein analogs to retard degradation by sample contaminants.

Suitable protein target analytes include, but are not limited to, (1)immunoglobulins, particularly IgEs, IgGs and IgMs, and particularlytherapeutically or diagnostically relevant antibodies, including but notlimited to, for example, antibodies to human albumin, apolipoproteins(including apolipoprotein E), human chorionic gonadotropin, cortisol,α-fetoprotein, thyroxin, thyroid stimulating hormone (TSH),antithrombin, antibodies to pharmaceuticals (including antieptilepticdrugs (phenyloin, primidone, carbariezepin, ethosuximide, valproic acid,and phenobarbitol), cardioactive drugs (digoxin, lidocaine,procainamide, and disopyramide), bronchodilators (theophylline),antibiotics (chloramphenicol, sulfonamides), antidepressants,immunosuppresants, abused drugs (amphetamine, methamphetamine,cannabinoids, cocaine and opiates) and antibodies to any number ofviruses (including orthomyxoviruses, (e.g. influenza virus),paramyxoviruses (e.g respiratory syncytial virus, mumps virus, measlesvirus), adenoviruses, rhinoviruses, coronaviruses, reoviruses,togaviruses (e.g. rubella virus), parvoviruses, poxviruses (e.g. variolavirus, vaccinia virus), enteroviruses (e.g. poliovirus, coxsackievirus),hepatitis viruses (including A, B and C), herpesviruses (e.g. Herpessimplex virus, varicella zoster virus, cytomegalovirus, Epstein Barrvirus), rotaviruses, Norwalk viruses, hantavirus, arenavirus,rhabdovirus (e.g. rabies virus), retroviruses (including HIV, HTLV I andII), papovaviruses (e.g. papillomavirus), polyomaviruses, andpicornaviruses, and the like), and bacteria (including a wide variety ofpathogenic and non pathogenic prokaryotes of interest includingBacillus; Vibrio, e.g. V. cholerae; Escherichia, e.g. Enterotoxigenic E.coli, Shigella, e.g. S. dysenteriae; Salmonella, e.g. S. typhi;Mycobacterium e.g. M. tuberculosis, M. leprae; Clostridium, e.g. C.botulinum, C. tetani, C. difficile, C. perfringens; Cornyebacterium,e.g. C. diphtheriae; Streptococcus, S. pyogenes, S. pneumoniae;Staphylococcus, e.g. S. aureus; Haemophilus, e.g. H. influenzae;Neisseria, e.g. N. meningitidis, N. gonorrhoeae; Yersinia, e.g. G.lamblia Y. pestis, Pseudomonas, e.g. P. aeruginosa, P. putida;Chlamydia, e.g. C. trachomatis; Bordetella, e.g. B. pertussis;Treponema, e.g. T. palladium; and the like); (2) enzymes (and otherproteins), including but not limited to, enzymes used as indicators ofor treatment for heart disease, including creatine kinase, lactatedehydrogenase, aspartate amino transferase, troponin T, myoglobin,fibrinogen, cholesterol, triglycerides, thrombin, tissue plasminogenactivator (tPA); pancreatic disease indicators including amylase,lipase, chymotrypsin and trypsin; liver function enzymes and proteinsincluding cholinesterase, bilirubin, and alkaline phosphotase; aldolase,prostatic acid phosphatase, terminal deoxynucleotidyl transferase, andbacterial and viral enzymes such as HIV protease; (3) hormones andcytokines (many of which serve as ligands for cellular receptors) suchas erythropoietin (EPO), thrombopoietin (TPO), the interleukins(including IL-1 through IL-17), insulin, insulin-like growth factors(including IGF-1 and -2), epidermal growth factor (EGF), transforminggrowth factors (including TGF-α and TGF-β), human growth hormone,transferrin, epidermal growth factor (EGF), low density lipoprotein,high density lipoprotein, leptin, VEGF, PDGF, ciliary neurotrophicfactor, prolactin, adrenocorticotropic hormone (ACTH), calcitonin, humanchorionic gonadotropin, cotrisol, estradiol, follicle stimulatinghormone (FSH), thyroid-stimulating hormone (TSH), leutinzing hormone(LH), progeterone, testosterone,; and (4) other proteins (includingα-fetoprotein, carcinoembryonic antigen CEA.

In addition, any of the biomolecules for which antibodies may bedetected may be detected directly as well; that is, detection of virusor bacterial cells, therapeutic and abused drugs, etc., may be donedirectly.

Suitable target analytes include carbohydrates, including but notlimited to, markers for breast cancer (CA15-3, CA 549, CA 27.29),mucin-like carcinoma associated antigen (MCA), ovarian cancer (CA125),pancreatic cancer (DE-PAN-2), and colorectal and pancreatic cancer (CA19, CA 50, CA242).

Target analytes including troponin and HbA1c find use in particularembodiments and applications. For HbA1c, one of the binding ligands,either the capture binding ligand or the soluble binding ligand hasspecificity for the glycated form of hemoglobin. That is, in oneembodiment, the capture binding ligand can bind either form ofhemoglobin; after washing the surface, a soluble binding ligand that hasspecificity only for the glycosylated form (e.g. HbA1c) with theperoxide-generating moiety is added. Alternatively, the capture bindingligand has specificity for Hb1Ac over other forms of hemoglobin, and asoluble capture ligand without such specificity can be used afterappropriate washing of the surface. This approach can be used for othertarget analytes where detection of either the glycosylated ornonglycosylated form is desired. As will be appreciated by those in theart, there are also target analytes for which detection of both forms isdesired, and in those embodiments, using binding ligands that do nothave specificity for one or the other is used.

Of particular interest in the present invention are assays forStaphylococcus enterotoxin B, P-Selectin, D-dimer, B-Type NatriureticPeptide (BNP), C Reactive Protein, Myoglobin and CK-MB.

In some embodiments, as outlined herein, any number of different enzymesubstrates, including metabolites, can serve as target analytes. Forexample, ATP and/or NADH, can be detected and/or quantified using thesystems herein. In addition, other enzyme substrates for whichperoxide-generating enzymes exist can be detected using the generalmechanisms as outlined herein, including, but not limited to, NADH,glucose, sucrose, lactose, lactate, galactose, glutamate, choline,ethanol, creatinine, etc.

Samples

The target analytes are generally present in samples. As will beappreciated by those in the art, the sample solution may comprise anynumber of things, including, but not limited to, bodily fluids(including, but not limited to, blood, urine, serum, lymph, saliva, analand vaginal secretions, perspiration and semen, of virtually anyorganism, with mammalian samples being preferred and human samples beingparticularly preferred); environmental samples (including, but notlimited to, air, agricultural, water and soil samples); plant materials;biological warfare agent samples; research samples, purified samples,raw samples, etc.; as will be appreciated by those in the art, virtuallyany experimental manipulation may have been done on the sample. Someembodiments utilize target samples from stored (e.g. frozen and/orarchived) or fresh tissues. Paraffin-embedded samples are of particularuse in many embodiments, as these samples can be very useful, due to thepresence of additional data associated with the samples, such asdiagnosis and prognosis. Fixed and paraffin-embedded tissue samples asdescribed herein refers to storable or archival tissue samples. Mostpatient-derived pathological samples are routinely fixed andparaffin-embedded to allow for histological analysis and subsequentarchival storage.

Solid Supports

The target analytes are detected using solid supports comprisingelectrodes. By “substrate” or “solid support” or other grammaticalequivalents herein is meant any material that can be modified to containdiscrete individual sites appropriate of the attachment or associationof capture ligands. Suitable substrates include metal surfaces such asgold, electrodes as defined below, glass and modified or functionalizedglass, fiberglass, teflon, ceramics, mica, plastic (including acrylics,polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyimide, polycarbonate,polyurethanes, Teflon™ and derivatives thereof, etc.), GETEK (a blend ofpolypropylene oxide and fiberglass), etc, polysaccharides, nylon ornitrocellulose, resins, silica or silica-based materials includingsilicon and modified silicon, carbon, metals, inorganic glasses and avariety of other polymers, with printed circuit board (PCB) materialsbeing particularly preferred.

The present system finds particular utility in array formats, i.e.wherein there is a matrix of addressable detection electrodes (hereingenerally referred to “pads”, “addresses” or “micro-locations”). By“array” herein is meant a plurality of capture ligands in an arrayformat; the size of the array will depend on the composition and end useof the array. Arrays containing from about 2 different capturesubstrates to many thousands can be made.

In a preferred embodiment, the detection electrodes are formed on asubstrate. In addition, the discussion herein is generally directed tothe use of gold electrodes, but as will be appreciated by those in theart, other electrodes can be used as well. The substrate can comprise awide variety of materials, as outlined herein and in the citedreferences.

In general, preferred materials include printed circuit board materials.Circuit board materials are those that comprise an insulating substratethat is coated with a conducting layer and processed using lithographytechniques, particularly photolithography techniques, to form thepatterns of electrodes and interconnects (sometimes referred to in theart as interconnections or leads). The insulating substrate isgenerally, but not always, a polymer. As is known in the art, one or aplurality of layers may be used, to make either “two dimensional” (e.g.all electrodes and interconnections in a plane) or “three dimensional”(wherein the electrodes are on one surface and the interconnects may gothrough the board to the other side or wherein electrodes are on aplurality of surfaces) boards. Three dimensional systems frequently relyon the use of drilling or etching, followed by electroplating with ametal such as copper, such that the “through board” interconnections aremade. Circuit board materials are often provided with a foil alreadyattached to the substrate, such as a copper foil, with additional copperadded as needed (for example for interconnections), for example byelectroplating. The copper surface may then need to be roughened, forexample through etching, to allow attachment of the adhesion layer.

Accordingly, in a preferred embodiment, the present invention providesbiochips (sometimes referred to herein “chips”) that comprise substratescomprising a plurality of electrodes, preferably gold electrodes. Thenumber of electrodes is as outlined for arrays. Each electrodepreferably comprises a self-assembled monolayer as outlined herein. In apreferred embodiment, one of the monolayer-forming species comprises acapture ligand as outlined herein. In addition, each electrode has aninterconnection that is attached to the electrode at one end and isultimately attached to a device that can control the electrode. That is,each electrode is independently addressable.

Finally, the compositions of the invention can include a wide variety ofadditional components, including microfluidic components and roboticcomponents (see for example U.S. Pat. Nos. 6,942,771 and 7,312,087 andrelated cases, both of which are hereby incorporated by reference in itsentirety), and detection systems including computers utilizing signalprocessing techniques (see for example U.S. Pat. No. 6,740,518, herebyincorporated by reference in its entirety).

Electrodes

The solid supports of the invention comprise electrodes. By “electrodes”herein is meant a composition, which, when connected to an electronicdevice, is able to sense a current or charge and convert it to a signal.Preferred electrodes are known in the art and include, but are notlimited to, certain metals and their oxides, including gold; platinum;palladium; silicon; aluminum; metal oxide electrodes including platinumoxide, titanium oxide, tin oxide, indium tin oxide, palladium oxide,silicon oxide, aluminum oxide, molybdenum oxide (Mo₂O₆), tungsten oxide(WO₃) and ruthenium oxides; and carbon (including glassy carbonelectrodes, graphite and carbon paste). Preferred electrodes includegold, silicon, carbon and metal oxide electrodes, with gold beingparticularly preferred.

The electrodes described herein are depicted as a flat surface, which isonly one of the possible conformations of the electrode and is forschematic purposes only. The conformation of the electrode will varywith the detection method used.

The electrodes of the invention are generally incorporated into biochipcartridges and can take a wide variety of configurations, and caninclude working and reference electrodes, interconnects (including“through board” interconnects), and microfluidic components. See forexample U.S. Pat. No. 7,312,087, incorporated herein by reference in itsentirety. In addition, the biochips generally include a workingelectrode with the components described herein, a reference electrode,and a counter/auxiliary electrode.

The biochip cartridges include substrates comprising the arrays ofbiomolecules, and can be configured in a variety of ways. For example,the chips can include reaction chambers with inlet and outlet ports forthe introduction and removal of reagents. In addition, the cartridgescan include caps or lids that have microfluidic components, such thatthe sample can be introduced, reagents added, reactions done, and thenthe sample is added to the reaction chamber comprising the array fordetection.

Self Assembled Monolayers

The electrodes comprise a self assembled monolayer (“SAM”). By“monolayer” or “self-assembled monolayer” or “SAM” herein is meant arelatively ordered assembly of molecules spontaneously chemisorbed on asurface, in which the molecules are oriented approximately parallel toeach other and roughly perpendicular to the surface. Each of themolecules includes a functional group that adheres to the surface, and aportion that interacts with neighboring molecules in the monolayer toform the relatively ordered array. A “mixed” monolayer comprises aheterogeneous monolayer, that is, where at least two different moleculesmake up the monolayer. As outlined herein, the use of a monolayerreduces the amount of non-specific binding of biomolecules to thesurface, and, in the case of nucleic acids, increases the efficiency ofoligonucleotide hybridization as a result of the distance of theoligonucleotide from the electrode. Thus, a monolayer facilitates themaintenance of the target enzyme away from the electrode surface. Inaddition, a monolayer serves to keep charge carriers away from thesurface of the electrode. Thus, this layer helps to prevent electricalcontact between the electrodes and the ReAMs, or between the electrodeand charged species within the solvent. Such contact can result in adirect “short circuit” or an indirect short circuit via charged specieswhich may be present in the sample. Accordingly, the monolayer ispreferably tightly packed in a uniform layer on the electrode surface,such that a minimum of “holes” exist. The monolayer thus serves as aphysical barrier to block solvent accessibility to the electrode.

In some embodiments, the monolayer comprises conductive oligomers, andin particular, conductive oligomers are generally used to attach the EAMto the electrode surface, as described below. By “conductive oligomer”herein is meant a substantially conducting oligomer, preferably linear,some embodiments of which are referred to in the literature as“molecular wires”. By “substantially conducting” herein is meant thatthe oligomer is capable of transferring electrons at 100 Hz. Generally,the conductive oligomer has substantially overlapping π-orbitals, i.e.conjugated π-orbitals, as between the monomeric units of the conductiveoligomer, although the conductive oligomer may also contain one or moresigma (σ) bonds. Additionally, a conductive oligomer may be definedfunctionally by its ability to inject or receive electrons into or froman associated EAM. Furthermore, the conductive oligomer is moreconductive than the insulators as defined herein. Additionally, theconductive oligomers of the invention are to be distinguished fromelectroactive polymers, that themselves may donate or accept electrons.

A more detailed description of conductive oligomers is found inWO/1999/57317, herein incorporated by reference in its entirety. Inparticular, the conductive oligomers as shown in Structures 1 to 9 onpage 14 to 21 of WO/1999/57317 find use in the present invention. Insome embodiments, the conductive oligomer has the following structure:

In addition, the terminus of at least some of the conductive oligomersin the monolayer is electronically exposed. By “electronically exposed”herein is meant that upon the placement of an EAM in close proximity tothe terminus, and after initiation with the appropriate signal, a signaldependent on the presence of the EAM may be detected. The conductiveoligomers may or may not have terminal groups. Thus, in a preferredembodiment, there is no additional terminal group, and the conductiveoligomer terminates with a terminal group; for example, such as anacetylene bond. Alternatively, in some embodiments, a terminal group isadded, sometimes depicted herein as “Q”. A terminal group may be usedfor several reasons; for example, to contribute to the electronicavailability of the conductive oligomer for detection of EAMs, or toalter the surface of the SAM for other reasons, for example to preventnon-specific binding. For example, there may be negatively chargedgroups on the terminus to form a negatively charged surface such thatwhen the target analyte is nucleic acid such as DNA or RNA, the nucleicacid is repelled or prevented from lying down on the surface, tofacilitate hybridization. Preferred terminal groups include —NH, —OH,—COOH, and alkyl groups such as —CH₃, and (poly)alkyloxides such as(poly)ethylene glycol, with —OCH₂CH₂OH, —(OCH₂CH₂O)₂H, —(OCH₂CH₂O)₃H,and —(OCH₂CH₂O)₄H being preferred.

In one embodiment, it is possible to use mixtures of conductiveoligomers with different types of terminal groups. Thus, for example,some of the terminal groups may facilitate detection, and some mayprevent non-specific binding.

In some embodiments, the electrode further comprises a passivationagent, preferably in the form of a monolayer on the electrode surface.For some analytes the efficiency of analyte binding (i.e. hybridization)may increase when the binding ligand is at a distance from theelectrode. In addition, the presence of a monolayer can decreasenon-specific binding to the surface (which can be further facilitated bythe use of a terminal group, outlined herein. A passivation agent layerfacilitates the maintenance of the binding ligand and/or analyte awayfrom the electrode surface. In addition, a passivation agent serves tokeep charge carriers away from the surface of the electrode. Thus, thislayer helps to prevent electrical contact between the electrodes and theelectron transfer moieties, or between the electrode and charged specieswithin the solvent. Such contact can result in a direct “short circuit”or an indirect short circuit via charged species which may be present inthe sample. Accordingly, the monolayer of passivation agents ispreferably tightly packed in a uniform layer on the electrode surface,such that a minimum of “holes” exist. Alternatively, the passivationagent may not be in the form of a monolayer, but may be present to helpthe packing of the conductive oligomers or other characteristics.

The passivation agents thus serve as a physical barrier to block solventaccessibility to the electrode. As such, the passivation agentsthemselves may in fact be either (1) conducting or (2) nonconducting,i.e. insulating, molecules. Thus, in one embodiment, the passivationagents are conductive oligomers, as described herein, with or without aterminal group to block or decrease the transfer of charge to theelectrode. Other passivation agents which may be conductive includeoligomers of —(CF₂)_(n)—, —(CHF)_(n)— and —(CFR)_(n)—. In a preferredembodiment, the passivation agents are insulator moieties.

In some embodiments, the monolayers comprise insulators. An “insulator”is a substantially nonconducting oligomer, preferably linear. By“substantially nonconducting” herein is meant that the rate of electrontransfer through the insulator is slower than the rate of electrontransfer through the conductive oligomer. Stated differently, theelectrical resistance of the insulator is higher than the electricalresistance of the conductive oligomer. It should be noted however thateven oligomers generally considered to be insulators, such as —(CH₂)₁₆molecules, still may transfer electrons, albeit at a slow rate.

In some embodiments, the insulators have a conductivity, S, of about10⁻⁷Ω⁻¹ cm⁻¹ or lower, with less than about 10⁻⁸Ω⁻¹ cm⁻¹ beingpreferred. Gardner et al., Sensors and Actuators A 51 (1995) 57-66,incorporated herein by reference.

Generally, insulators are alkyl or heteroalkyl oligomers or moietieswith sigma bonds, although any particular insulator molecule may containaromatic groups or one or more conjugated bonds. By “heteroalkyl” hereinis meant an alkyl group that has at least one heteroatom, i.e. nitrogen,oxygen, sulfur, phosphorus, silicon or boron included in the chain.Alternatively, the insulator may be quite similar to a conductiveoligomer with the addition of one or more heteroatoms or bonds thatserve to inhibit or slow, preferably substantially, electron transfer.In some embodiments the insulator comprises C₆-C₁₆ alkyl.

The passivation agents, including insulators, may be substituted with Rgroups as defined herein to alter the packing of the moieties orconductive oligomers on an electrode, the hydrophilicity orhydrophobicity of the insulator, and the flexibility, i.e. therotational, torsional or longitudinal flexibility of the insulator. Forexample, branched alkyl groups may be used. In addition, the terminus ofthe passivation agent, including insulators, may contain an additionalgroup to influence the exposed surface of the monolayer, sometimesreferred to herein as a terminal group (“TG”). For example, the additionof charged, neutral or hydrophobic groups may be done to inhibitnon-specific binding from the sample, or to influence the kinetics ofbinding of the analyte, etc. For example, there may be charged groups onthe terminus to form a charged surface to encourage or discouragebinding of certain target analytes or to repel or prevent from lyingdown on the surface.

The length of the passivation agent will vary as needed. Generally, thelength of the passivation agents is similar to the length of theconductive oligomers, as outlined above. In addition, the conductiveoligomers may be basically the same length as the passivation agents orlonger than them, resulting in the binding ligands being more accessibleto the solvent.

The monolayer may comprise a single type of passivation agent, includinginsulators, or different types.

Suitable insulators are known in the art, and include, but are notlimited to, —(CH₂)_(n)—, —(CRH)_(n)—, and —(CR₂)_(n)—, ethylene glycolor derivatives using other heteroatoms in place of oxygen, i.e. nitrogenor sulfur (sulfur derivatives are not preferred when the electrode isgold). In some embodiments, the insulator comprises C₆ to C₁₆ alkyl.

In some embodiments, the electrode is a metal surface and need notnecessarily have interconnects or the ability to do electrochemistry.

Electroactive Moieties

In addition to the SAMs, the electrodes comprise an EAM. By“electroactive moiety (EAM)” or “transition metal complex” or “redoxactive molecule” or “electron transfer moiety (ETM)” herein is meant ametal-containing compound which is capable of reversibly orsemi-reversibly transferring one or more electrons. It is to beunderstood that electron donor and acceptor capabilities are relative;that is, a molecule which can lose an electron under certainexperimental conditions will be able to accept an electron underdifferent experimental conditions.

It is to be understood that the number of possible transition metalcomplexes is very large, and that one skilled in the art of electrontransfer compounds will be able to utilize a number of compounds in thepresent invention. By “transitional metal” herein is meant metals whoseatoms have a partial or completed shell of electrons. Suitabletransition metals for use in the invention include, but are not limitedto, cadmium (Cd), copper (Cu), cobalt (Co), palladium (Pd), zinc (Zn),iron (Fe), ruthenium (Ru), rhodium (Rh), osmium (Os), rhenium (Re),platinum (Pt), scandium (Sc), titanium (Ti), Vanadium (V), chromium(Cr), manganese (Mn), nickel (Ni), Molybdenum (Mo), technetium (Tc),tungsten (W), and iridium (Ir). That is, the first series of transitionmetals, the platinum metals (Ru, Rh, Pd, Os, Ir and Pt), along with Fe,Re, W, Mo and Tc, find particular use in the present invention. Metalsthat find use in the invention also are those that do not change thenumber of coordination sites upon a change in oxidation state, includingruthenium, osmium, iron, platinum and palladium, with osmium, rutheniumand iron being especially useful. Generally, transition metals aredepicted herein (or in incorporated references) as “TM” or “M”.

The transitional metal and the coordinating ligands form a metalcomplex. By “ligand” or “coordinating ligand” (depicted herein or inincorporated references in the figures as “L”) herein is meant an atom,ion, molecule, or functional group that generally donates one or more ofits electrons through a coordinate covalent bond to, or shares itselectrons through a covalent bond with, one or more central atoms orions.

In some embodiments, small polar ligands are used; suitable small polarligands, generally depicted herein as “L”, fall into two generalcategories, as is more fully described herein. In one embodiment, thesmall polar ligands will be effectively irreversibly bound to the metalion, due to their characteristics as generally poor leaving groups or asgood sigma donors, and the identity of the metal. These ligands may bereferred to as “substitutionally inert”. Alternatively, as is more fullydescribed below, the small polar ligands may be reversibly bound to themetal ion, such that upon binding of a target analyte, the analyte mayprovide one or more coordination atoms for the metal, effectivelyreplacing the small polar ligands, due to their good leaving groupproperties or poor sigma donor properties. These ligands may be referredto as “substitutionally labile”. The ligands preferably form dipoles,since this will contribute to a high solvent reorganization energy.

Some of the structures of transitional metal complexes are shown below:

L are the co-ligands, that provide the coordination atoms for thebinding of the metal ion. As will be appreciated by those in the art,the number and nature of the co-ligands will depend on the coordinationnumber of the metal ion. Mono-, di- or polydentate co-ligands may beused at any position. Thus, for example, when the metal has acoordination number of six, the L from the terminus of the conductiveoligomer, the L contributed from the nucleic acid, and r, add up to six.Thus, when the metal has a coordination number of six, r may range fromzero (when all coordination atoms are provided by the other two ligands)to four, when all the co-ligands are monodentate. Thus generally, r willbe from 0 to 8, depending on the coordination number of the metal ionand the choice of the other ligands.

In one embodiment, the metal ion has a coordination number of six andboth the ligand attached to the conductive oligomer and the ligandattached to the nucleic acid are at least bidentate; that is, r ispreferably zero, one (i.e. the remaining co-ligand is bidentate) or two(two monodentate co-ligands are used).

As will be appreciated in the art, the co-ligands can be the same ordifferent. Suitable ligands fall into two categories: ligands which usenitrogen, oxygen, sulfur, carbon or phosphorus atoms (depending on themetal ion) as the coordination atoms (generally referred to in theliterature as sigma (σ) donors) and organometallic ligands such asmetallocene ligands (generally referred to in the literature as pi (π)donors, and depicted herein as L_(m)). Suitable nitrogen donatingligands are well known in the art and include, but are not limited to,cyano (C≡N), NH₂; NHR; NRR′; pyridine; pyrazine; isonicotinamide;imidazole; bipyridine and substituted derivatives of bipyridine;terpyridine and substituted derivatives; phenanthrolines, particularly1,10-phenanthroline (abbreviated phen) and substituted derivatives ofphenanthrolines such as 4,7-dimethylphenanthroline anddipyridol[3,2-a:2′,3′-c]phenazine (abbreviated dppz); dipyridophenazine;1,4,5,8,9,12-hexaazatriphenylene (abbreviated hat);9,10-phenanthrenequinone diimine (abbreviated phi);1,4,5,8-tetraazaphenanthrene (abbreviated tap);1,4,8,11-tetra-azacyclotetradecane (abbreviated cyclam) and isocyanide.Substituted derivatives, including fused derivatives, may also be used.In some embodiments, porphyrins and substituted derivatives of theporphyrin family may be used. See for example, ComprehensiveCoordination Chemistry, Ed. Wilkinson et al., Pergammon Press, 1987,Chapters 13.2 (pp 73-98), 21.1 (pp. 813-898) and 21.3 (pp 915-957), allof which are hereby expressly incorporated by reference.

As will be appreciated in the art, any ligand donor(1)-bridge-donor(2)where donor (1) binds to the metal and donor(2) is available forinteraction with the surrounding medium (solvent, protein, etc.) can beused in the present invention, especially if donor(1) and donor(2) arecoupled through a pi system, as in cyanos (C is donor(1), N is donor(2),pi system is the CN triple bond). One example is bipyrimidine, whichlooks much like bipyridine but has N donors on the “back side” forinteractions with the medium. Additional co-ligands include, but are notlimited to cyanates, isocyanates (—N═C═O), thiocyanates, isonitrile, N₂,O₂, carbonyl, halides, alkoxyide, thiolates, amides, phosphides, andsulfur containing compound such as sulfino, sulfonyl, sulfoamino, andsulfamoyl.

In some embodiments, multiple cyanos are used as co-ligand to complexwith different metals. For example, seven cyanos bind Re(III); eightbind Mo(IV) and W(IV). Thus at Re(III) with 6 or less cyanos and one ormore L, or Mo(IV) or W(IV) with 7 or less cyanos and one or more L canbe used in the present invention. The EAM with W(IV) system hasparticular advantages over the others because it is more inert, easierto prepare, more favorable reduction potential. Generally that a largerCN/L ratio will give larger shifts.

Suitable sigma donating ligands using carbon, oxygen, sulfur andphosphorus are known in the art. For example, suitable sigma carbondonors are found in Cotton and Wilkinson, Advanced Organic Chemistry,5th Edition, John Wiley & Sons, 1988, hereby incorporated by reference;see page 38, for example. Similarly, suitable oxygen ligands includecrown ethers, water and others known in the art. Phosphines andsubstituted phosphines are also suitable; see page 38 of Cotton andWilkinson.

The oxygen, sulfur, phosphorus and nitrogen-donating ligands areattached in such a manner as to allow the heteroatoms to serve ascoordination atoms.

In some embodiments, organometallic ligands are used. In addition topurely organic compounds for use as redox moieties, and varioustransition metal coordination complexes with 5-bonded organic ligandwith donor atoms as heterocyclic or exocyclic substituents, there isavailable a wide variety of transition metal organometallic compoundswith π-bonded organic ligands (see Cotton & Wilkinson, chapter 26;Organometallics, A Concise Introduction, Elschenbroich et al., 2nd Ed.,1992, VCH; and Comprehensive Organometallic Chemistry II, A Review ofthe Literature 1982-1994, Abel et al. Ed., Vol. 7, chapters 7, 8, 10 &11, Pergamon Press, hereby expressly incorporated by reference). Suchorganometallic ligands include cyclic aromatic compounds such as thecyclopentadienide ion [C₅H₅]⁽⁻¹⁾ and various ring substituted and ringfused derivatives, such as the indenylide (−1) ion, that yield a classof bis(cyclopentadieyl)metal compounds, (i.e. the metallocenes); see forexample Robins et al., J. Am. Chem. Soc. 104:1882-1893 (1982); andGassman et al., J. Am. Chem. Soc. 108:4228-4229 (1986), incorporated byreference. Of these, ferrocene [(C₅H₅)₂Fe] and its derivatives areprototypical examples which have been used in a wide variety of chemical(Connelly et al., Chem. Rev. 96:877-910 (1996), incorporated byreference) and electrochemical (Geiger et al., Advances inOrganometallic Chemistry 23:1-93; and Geiger et al., Advances inOrganometallic Chemistry 24:87, incorporated by reference) electrontransfer or “redox” reactions. Metallocene derivatives of a variety ofthe first, second and third row transition metals are potentialcandidates as redox moieties that are covalently attached to either theribose ring or the nucleoside base of nucleic acid. Other potentiallysuitable organometallic ligands include cyclic arenes such as benzene,to yield bis(arene)metal compounds and their ring substituted and ringfused derivatives, of which bis(benzene)chromium is a prototypicalexample. Other acyclic π-bonded ligands such as the allyl(−1) ion, orbutadiene yield potentially suitable organometallic compounds, and allsuch ligands, in conduction with other .pi.-bonded and .delta.-bondedligands constitute the general class of organometallic compounds inwhich there is a metal to carbon bond. Electrochemical studies ofvarious dimers and oligomers of such compounds with bridging organicligands, and additional non-bridging ligands, as well as with andwithout metal-metal bonds are potential candidate redox moieties innucleic acid analysis.

When one or more of the co-ligands is an organometallic ligand, theligand is generally attached via one of the carbon atoms of theorganometallic ligand, although attachment may be via other atoms forheterocyclic ligands. Preferred organometallic ligands includemetallocene ligands, including substituted derivatives and themetalloceneophanes (see page 1174 of Cotton and Wilkinson, supra). Forexample, derivatives of metallocene ligands such asmethylcyclopentadienyl, with multiple methyl groups being preferred,such as pentamethylcyclopentadienyl, can be used to increase thestability of the metallocene. In a preferred embodiment, only one of thetwo metallocene ligands of a metallocene are derivatized.

As described herein, any combination of ligands may be used. Preferredcombinations include: a) all ligands are nitrogen donating ligands; b)all ligands are organometallic ligands; and c) the ligand at theterminus of the conductive oligomer is a metallocene ligand and theligand provided by the nucleic acid is a nitrogen donating ligand, withthe other ligands, if needed, are either nitrogen donating ligands ormetallocene ligands, or a mixture.

As a general rule, EAM comprising non-macrocyclic chelators are bound tometal ions to form non-macrocyclic chelate compounds, since the presenceof the metal allows for multiple proligands to bind together to givemultiple oxidation states.

In some embodiments, nitrogen donating proligands are used. Suitablenitrogen donating proligands are well known in the art and include, butare not limited to, NH₂; NHR; NRR′; pyridine; pyrazine; isonicotinamide;imidazole; bipyridine and substituted derivatives of bipyridine;terpyridine and substituted derivatives; phenanthrolines, particularly1,10-phenanthroline (abbreviated phen) and substituted derivatives ofphenanthrolines such as 4,7-dimethylphenanthroline anddipyridol[3,2-a:2′,3′-c]phenazine (abbreviated dppz); dipyridophenazine;1,4,5,8,9,12-hexaazatriphenylene (abbreviated hat);9,10-phenanthrenequinone diimine (abbreviated phi);1,4,5,8-tetraazaphenanthrene (abbreviated tap);1,4,8,11-tetra-azacyclotetradecane (abbreviated cyclam) and isocyanide.Substituted derivatives, including fused derivatives, may also be used.It should be noted that macrocylic ligands that do not coordinativelysaturate the metal ion, and which require the addition of anotherproligand, are considered non-macrocyclic for this purpose. As will beappreciated by those in the art, it is possible to covalent attach anumber of “non-macrocyclic” ligands to form a coordinatively saturatedcompound, but that is lacking a cyclic skeleton.

In some embodiments, a mixture of monodentate (e.g. at least one cyanoligand), bi-dentate, tri-dentate, and polydentate ligands can be used inthe construction of EAMs.

Of particular use in the present invention are EAMs that aremetallocenes, and in particular ferrocenes, which have at least a firstself-immolative moiety attached, although in some embodiments, more thanone self-immolative moiety is attached as is described below (it shouldalso be noted that other EAMs, as are broadly described herein, withself-immolative moieties can also be used). In some embodiments, whenmore than one self-immolative moiety is attached to a ferrocene, theyare all attached to one of the cyclopentydienyl rings. In someembodiments, the self-immolative moieties are attached to differentrings. In some embodiments, it is possible to saturate one or both ofthe cyclopentydienyl rings with self-immolative moieties, as long as onesite is used for attachment to the electrode.

In addition, EAMs generally have an attachment moiety for attachment ofthe EAM to the conductive oligomer which is used to attach the EAM tothe electrode. In general, although not required, in the case ofmetallocenes such as ferrocenes, the self-immolative moiety(ies) areattached to one of the cyclopentydienyl rings, and the attachment moietyis attached to the other ring, as is generally depicted in FIG. 1,although attachment to the same ring can also be done. As will beappreciated by those in the art, any combination of self-immolativemoieties and at least one attachment linker can be used, and on eitherring.

In addition to the self-immolative moiety(ies) and the attachmentmoiety(ies), the ferrocene can comprise additional substituent groups,which can be added for a variety of reasons, including altering the E⁰in the presence or absence of at least the self-immolative group.Suitable substituent groups, frequently depicted in associated andincorporated references as “R” groups, are recited in U.S. patentapplication Ser. No. 12/253,828, filed Oct. 17, 2008; U.S. patentapplication Ser. No. 12/253,875, filed Oct. 17, 2008; U.S. ProvisionalPatent Application No. 61/332,565, filed May 7, 2010; U.S. ProvisionalPatent Application No. 61/347,121, filed May 21, 2010; and U.S.Provisional Patent Application No. 61/366,013, filed Jul. 20, 2010,hereby incorporated by reference.

In some embodiments, such as depicted below, the EAM does not comprise aself-immolative moiety, in the case where the peroxide-sensitive moietyis attached directly to the EAM and provides a change in E⁰ when theperoxide-sensitive moiety is exposed to peroxide. As shown below, oneembodiment allows the peroxide-sensitive moiety to be attached directlyto the EAM (in this case, a ferrocene), such that the ferrocene has afirst E⁰ when the pinacol boronate ester moiety is attached, and asecond E⁰ when removed, e.g. in the presence of the peroxide.

Self-Immolative Moieties

The EAMs of the invention include at least one self-immolative moietythat is covalently attached to the EAM such that the EAM has a first E⁰when it is present and a second E⁰ when it has been removed as describedbelow.

The term “self-immolative spacer” refers to a bifunctional chemicalmoiety that is capable of covalently linking two chemical moieties intoa normally stable tripartate molecule. The self-immolative spacer iscapable of spontaneously separating from the second moiety if the bondto the first moiety is cleaved. In the present invention, theself-immolative spacer links a peroxide sensitive moiety, e.g. a boronmoiety, to the EAM. Upon exposure to peroxide, the boron moiety isremoved and the spacer falls apart, as generally depicted in FIG. 1.Generally speaking, any spacer where irreversible repetitive bondrearrangement reactions are initiated by an electron-donating alcoholfunctional group (i.e. quinone methide motifs) can be designed withboron groups serving as triggering moieties that generate alcohols underoxidative conditions. Alternatively, the boron moiety can mask a latentphenolic oxygen in a ligand that is a pro-chelator for a transitionmetal. Upon oxidation, the ligand is transformed and initiates EAMformation in the SAM. For example, a sample chelating ligand issalicaldehyde isonicotinoyl hydrazone that binds iron.

As will be appreciated by those in the art, a wide variety ofself-immolative moieties may be used with a wide variety of EAMs andperoxide sensitive moieties. Self-immolative linkers have been describedin a number of references, including US Publication Nos. 20090041791;20100145036 and U.S. Pat. Nos. 7,705,045 and 7,223,837, all of which areexpressly incorporated by reference in their entirety, particularly forthe disclosure of self-immolative spacers.

A few self-immolative linkers of particular use in the present inventionare shown in FIG. 6. The self-immolative spacer can comprise a singlemonomeric unit or polymers, either of the same monomers (homopolymers)or of different monomers (heteropolymers). Alternatively, theself-immolative spacer can be a neighboring group to an EAM in a SAMthat changes the environment of the EAM following cleavage analogous tothe chemistry as recited in previous application “Electrochemical Assayfor the Detection of Enzymes”, U.S. Ser. No. 12/253,828,PCT/US2008/080363, hereby incorporated by reference.

Peroxide Sensitive Moieties

The self-immolative spacers join the peroxide sensitive moieties (PSMs,sometimes referred to herein as POMs) and the EAMs of the invention. Ingeneral, a peroxide sensitive moiety is a moiety containing boron asdepicted in FIG. 1.

For example, the figures herein depict the use of ferrocene derivatives,where the peroxide triggers a change from a benzyl carbamate with ap-substituted pinacol borate ester to an amine. This self-eliminatinggroup has been described previously for generating amine-functionalizedflorophores in the presence of hydrogen peroxide (Sella, E.; Shabat, D.Self-immolative dendritic probe for the direct detection of triacetonetriperoxide. Chem. Commun. 2008, 5701-5703; and Lo, L.-Cl; Chu, C.-Y.Development of highly selective and sensitive probes for hydrogenperoxide. Chem. Commun. 2003, 2728-2729 both of which are incorporatedby reference. Other such groups (aryl borate esters and arylboronicacids) are also described in Sella and Lo. In addition, ferrocenylaminesare known to exhibit redox behavior at lower potentials (˜150 mV) ascompared to their corresponding carbamate derviatives (see Sagi et al.,Amperometric Assay for Aldolase Activity; Antibody-CatalyzedFerrocenylamine Formation. Anal. Chem. 2006, 78, 1459-1461, incorporatedby reference herein).

Capture and Soluble Binding Ligands

In addition to SAMs and EAMs, in some embodiments, the electrodescomprise capture binding ligands. By “binding ligand” or “bindingspecies” herein is meant a compound that is used to probe for thepresence of the target analyte and that will bind to the target analyte.In general, for most of the embodiments described herein, there are atleast two binding ligands used per target analyte molecule; a “capture”or “anchor” binding ligand that is attached to the detection surface,and a soluble binding ligand, that binds independently to the targetanalyte, and either directly or indirectly comprises at least one labelsuch as a SOX. By “capture binding ligand” herein is meant a bindingligand that binds the target analyte that is attached to the electrodesurface that binds the target analyte. By “soluble binding ligand”herein is meant a binding ligand that is in solution that binds thetarget analyte at a different site than the capture binding ligand.

As will be appreciated by those in the art, the composition of thebinding ligand will depend on the composition of the target analyte.Binding ligands for a wide variety of analytes are known or can bereadily found using known techniques. For example, when the analyte is aprotein, the binding ligands include proteins (particularly includingantibodies or fragments thereof (FAbs, etc.)) or small molecules.

In general, antibodies are useful as both capture and soluble bindingligands.

The soluble binding ligand also comprises a peroxide generating moietysuch as an enzyme that generates peroxide. A wide variety of suchenzymes are known, including glucose oxidase, acyl CoA oxidases, alcoholoxidases, aldehyde oxidases, etc. A wide variety of suitable oxidaseenzymes are known in the art (see any glucose oxidase enzyme classifiedas EC 1.1.3.4, including, but not limited to, glucose oxidase, D-aminoacid oxidase (DAAO) and choline oxidase). Glucose oxidase enzymes from awide variety of organisms are well known, including bacterial, fungaland animal (including mammalian), including, but not limited to,Aspergillus species (e.g. A. niger), Penicillum species, Streptomycesspecies, mouse, etc.). Also of use are acyl CoA oxidases, classified asEC 1.3.3.6.

Alternatively, the soluble binding ligand may contain an enzyme, such asalkaline phosphatase (AP), that catalyzes the generation of a necessarycofactor from a phosphorylated precursor for a soluble apo-oxidaseenzyme (i.e. FADP converted to FAD which binds to apo-DAAO) which inturn generates peroxide by reaction with substrate. This strategyenables cascade amplification of target binding events if theconcentrations of apo-enzyme, phosphorylated cofactor, and oxidaseenzyme substrate are high with respect to the surface immobilizedtarget.

Generally, the capture binding ligand allows the attachment of a targetanalyte to the detection surface, for the purposes of detection. In oneembodiment, the binding is specific, and the binding ligand is part of aspecific binding pair.

The term “specific binding pair” as used herein refers to two compoundsthat specifically bind to one another in a non-covalent manner, such asbut not limited to a receptor (e.g., enzyme) and a ligand; an antibodyand an antigen; complementary nucleic acids; or an aptamer and itstarget. A “first member of a specific binding pair” can be eitherelement of the binding pair (e.g., biotin or avoiding) and the “secondmember of the specific binding pair” can be the remaining element of thebinding pair (e.g., where the first member of a specific binding pair isbiotin, the second member of the specific binding pair can be avidin).

For example, the specific binding pair can be biotin and avidin orbiotin and streptavidin, or analogs thereof (i.e. biotin oravidin/streptavidin molecules that have been modified but yet a/low forreversible binding as described herein). In another example, thespecific binding pair can be an antigen and an antibody. Suitableantigens include, but are not limited to, fluorescein, biotin,digoxigenin, or dinitrophenol. In a further example, the specificbinding pair can also be an aptamer and its target molecule. Aptamerscan be short nucleic acid or short peptides (e.g., 6-40 kDa) whichstrongly bind a target molecule, typically with binding constants(K_(D)) in the micromolar to nanomolar range (i.e., <1000 μM to <1000nM). Aptamer targets can include, but are not limited to, an organic dye(e.g., fluorescein, Cy3, Cy5), a disaccharide (e.g., cellobiose,lactose, maltose, gentiobiose), an aminoglycoside (e.g., tobramycin,lividomycin, kanamycin A, kanamycin B, neomycin B), an antibiotic (e.g,viomycin and tetracyclin), dopamine, porphyrins (e.g., hematoporphyrin),and biotin.

“Nucleic acids” may be any natural or synthetic nucleic acids, includingDNA and RNA, and can be from 10 to 1,000 nucleotides in length. Incertain embodiments, the nucleic acids are 10 to 100 nucleotides inlength. In certain embodiments, the nucleic acids are 10 to 75nucleotides in length; or 10 to 50 nucleotides; or 10 to 40 nucleotidesin length. Shorter oligomers can be less costly but may not be robust;longer oligomers can be used for higher operating temperatures, or inharsher (e.g., pH or high salt concentration) environments.

For example, the specific binding pair can be complementary nucleicacids, such as two complementary single-stranded DNA molecules capableof forming duplex DNA, two complementary single-stranded RNA moleculescapable of forming double-stranded RNA, or a single-stranded DNAmolecule and a single-stranded RNA molecule capable of forming a DNA-RNAhybrid. It will be understood by one of skill in the art that the twoindividual nucleic acid molecules can form a binding pair complex underthe appropriate hybridization or annealing conditions, and that suchconditions can be optimized for the particular nucleic acid molecules atissue. It will be further understood by one of skill in the art thatonce formed, the duplex DNA, double-stranded RNA, or DNA-RNA hybrid canbe disassociated under appropriate denaturation conditions.

By “specifically bind” herein is meant that the ligand binds theanalyte, with specificity sufficient to differentiate between theanalyte and other components or contaminants of the test sample. Thebinding should be sufficient to allow the analyte to remain bound underthe conditions of the assay, including wash steps to remove non-specificbinding. In some embodiments, for example in the detection of certainbiomolecules, the binding constants of the analyte to the binding ligandwill be at least about 10⁻⁴ to 10⁻⁹ M⁻¹, with at least about 10⁻⁵ to10⁻⁹ being preferred and at least about 10⁻⁷ to 10⁻⁹ M⁻¹ beingparticularly preferred.

Binding ligands to a wide variety of analytes are known or can bereadily found using known techniques. For example, when the analyte is asingle-stranded nucleic acid, the binding ligand is generally asubstantially complementary nucleic acid. Alternatively, as is generallydescribed in U.S. Pat. Nos. 5,270,163, 5,475,096, 5,567,588, 5,595,877,5,637,459, 5,683,867, 5,705,337, and related patents, herebyincorporated by reference, nucleic acid “aptamers” can be developed forbinding to virtually any target analyte. Similarly the analyte may be anucleic acid binding protein and the capture binding ligand is either asingle-stranded or double-stranded nucleic acid; alternatively, thebinding ligand may be a nucleic acid binding protein when the analyte isa single or double-stranded nucleic acid. When the analyte is a protein,the binding ligands include proteins (particularly including antibodiesor fragments thereof (FAbs, etc.)), small molecules, or aptamers,described above. Preferred binding ligand proteins include antibodiesand peptides. As will be appreciated by those in the art, any twomolecules that will associate, preferably specifically, may be used,either as the analyte or the binding ligand. Suitable analyte/bindingligand pairs include, but are not limited to, antibodies/antigens,receptors/ligand, proteins/nucleic acids; nucleic acids/nucleic acids,enzymes/substrates and/or inhibitors, carbohydrates (includingglycoproteins and glycolipids)/lectins, carbohydrates and other bindingpartners, proteins/proteins; and protein/small molecules. These may bewild-type or derivative sequences.

The capture binding ligands (e.g. a capture antibody) can be covalentlycoupled to the electrode (usually through an attachment linker) or boundtightly but not covalently; for example, using biotin/streptavidinreactions (e.g. biotin on the surface of the SAM, streptavin-conjugatedcapture ligand such as an antibody, or vice versa), bound via a nucleicacid reaction (for example, the capture ligand can have a nucleic acid(“Watson”) and the surface can have a complementary nucleic acid(“Crick”), bound using protein G binding to the Fc fragment of theantibody, etc.

It should also be noted that the invention described herein can also beused as a sensor for the illicit explosive triacetone triperoxide(TATP).

Anchor Groups

The present invention provides compounds including the EAM (optionallyattached to the electrode surface with a conductive oligomer), the SAM,and the capture binding ligands on the electrode surface. Generally, insome embodiments, these moieties are attached to the electrode usinganchor group. By “anchor” or “anchor group” herein is meant a chemicalgroup that attaches the compounds of the invention to an electrode.

As will be appreciated by those in the art, the composition of theanchor group will vary depending on the composition of the surface towhich it is attached. In the case of gold electrodes, both pyridinylanchor groups and thiol based anchor groups find particular use.

The covalent attachment of the conductive oligomer may be accomplishedin a variety of ways, depending on the electrode and the conductiveoligomer used. Generally, some type of linker is used, as depicted belowas “A” in Structure 1, where X is the conductive oligomer, and thehatched surface is the electrode:

In this embodiment, A is a linker or atom. The choice of “A” will dependin part on the characteristics of the electrode. Thus, for example, Amay be a sulfur moiety when a gold electrode is used. Alternatively,when metal oxide electrodes are used, A may be a silicon (silane) moietyattached to the oxygen of the oxide (see for example Chen et al.,Langmuir 10:3332-3337 (1994); Lenhard et al., J. Electroanal. Chem.78:195-201 (1977), both of which are expressly incorporated byreference). When carbon based electrodes are used, A may be an aminomoiety (preferably a primary amine; see for example Deinhammer et al.,Langmuir 10:1306-1313 (1994)). Thus, preferred A moieties include, butare not limited to, silane moieties, sulfur moieties (including alkylsulfur moieties), and amino moieties.

In some embodiments, the electrode is a carbon electrode, i.e. a glassycarbon electrode, and attachment is via a nitrogen of an amine group. Arepresentative structure is depicted in Structure 15 of US PatentApplication Publication No. 20080248592, hereby incorporated byreference in its entirety but particularly for Structures as describedtherein and the description of different anchor groups and theaccompanying text. Again, additional atoms may be present, i.e. linkersand/or terminal groups.

In Structure 16 of US Patent Application Publication No. 20080248592,hereby incorporated by reference as above, the oxygen atom is from theoxide of the metal oxide electrode. The Si atom may also contain otheratoms, i.e. be a silicon moiety containing substitution groups. Otherattachments for SAMs to other electrodes are known in the art; see forexample Napier et al., Langmuir, 1997, for attachment to indium tinoxide electrodes, and also the chemisorption of phosphates to an indiumtin oxide electrode (talk by H. Holden Thorpe, CHI conference, May 4-5,1998).

In one preferred embodiment, indium-tin-oxide (ITO) is used as theelectrode, and the anchor groups are phosphonate-containing species.

1). Sulfur Anchor Groups

Although depicted in Structure 1 as a single moiety, the conductiveoligomer may be attached to the electrode with more than one “A” moiety;the “A” moieties may be the same or different. Thus, for example, whenthe electrode is a gold electrode, and “A” is a sulfur atom or moiety,multiple sulfur atoms may be used to attach the conductive oligomer tothe electrode, such as is generally depicted below in Structures 2, 3and 4. As will be appreciated by those in the art, other such structurescan be made. In Structures 2, 3 and 4 the A moiety is just a sulfuratom, but substituted sulfur moieties may also be used.

Thus, for example, when the electrode is a gold electrode, and “A” is asulfur atom or moiety, such as generally depicted below in Structure 6,multiple sulfur atoms may be used to attach the conductive oligomer tothe electrode, such as is generally depicted below in Structures 2, 3and 4. As will be appreciated by those in the art, other such structurescan be made. In Structures 2, 3 and 4, the A moiety is just a sulfuratom, but substituted sulfur moieties may also be used.

It should also be noted that similar to Structure 4, it may be possibleto have a conductive oligomer terminating in a single carbon atom withthree sulfur moieties attached to the electrode.

In another aspect, the present invention provide anchor compriseconjugated thiols. Some exemplary complexes with conjugated thiolanchors are shown in FIG. 10. In some embodiments, the anchor comprisesan alkylthiol group. Some of the examples are shown in FIGS. 10A and 4B.The two compounds depicts in FIG. 10B are based on carbene and4-pyridylalanine, respectively.

In another aspect, the present invention provides conjugated multipodalthio-containing compounds that serve as anchoring groups in theconstruction of electroactive moieties for analyte detection onelectrodes, such as gold electrodes. That is, spacer groups (which canbe attached to EAMs, ReAMCs, or an “empty” monolayer forming species)are attached using two or more sulfur atoms. These mulitpodal anchorgroups can be linear or cyclic, as described herein.

In some embodiments, the anchor groups are “bipodal”, containing twosulfur atoms that will attach to the gold surface, and linear, althoughin some cases it can be possible to include systems with othermultipodalities (e.g. “tripodal”). Such a multipodal anchoring groupdisplay increased stability and/or allow a greater footprint forpreparing SAMs from thiol-containing anchors with sterically demandingheadgroups.

In some embodiments, the anchor comprises cyclic disulfides (“bipod”).Although in some cases it can be possible to include ring system anchorgroups with other multipodalities (e.g. “tripodal”). The number of theatoms of the ring can vary, for example from 5 to 10, and also includesmulticyclic anchor groups, as discussed below

In some embodiments, the anchor groups comprise a [1,2,5]-dithiazepaneunit which is seven-membered ring with an apex nitrogen atom and aintramolecular disulfide bond as shown below:

In Structure (IIIa), it should also be noted that the carbon atoms ofthe ring can additionally be substituted. As will be appreciated bythose in the art, other membered rings are also included. In addition,multicyclic ring structures can be used, which can include cyclicheteroalkanes such as the [1,2,5]-dithiazepane shown above substitutedwith other cyclic alkanes (including cyclic heteroalkanes) or aromaticring structures.

In some embodiments, the anchor group and part of the spacer has thestructure shown below

The “R” group herein can be any substitution group, including aconjugated oligophenylethynylene unit with terminal coordinating ligandfor the transition metal component of the EAM.

The anchors are synthesized from a bipodal intermediate (I) (thecompound as formula III where R═I), which is described in Li et al.,Org. Lett. 4:3631-3634 (2002), herein incorporated by reference. Seealso Wei et al, J. Org, Chem. 69:1461-1469 (2004), herein incorporatedby reference.

The number of sulfur atoms can vary as outlined herein, with particularembodiments utilizing one, two, and three per spacer.

As will be appreciated by those in the art, the compositions of theinvention can be made in a variety of ways, including those outlinedbelow and in U.S. patent application Ser. No. 12/253,828, filed Oct. 17,2008; U.S. patent application Ser. No. 12/253,875, filed Oct. 17, 2008;U.S. Provisional Patent Application No. 61/332,565, filed May 7, 2010;U.S. Provisional Patent Application No. 61/347,121, filed May 21, 2010;U.S. Provisional Patent Application No. 61/366,013, filed Jul. 20, 2010.In some embodiments, the composition are made according to methodsdisclosed in of U.S. Pat. Nos. 6,013,459, 6,248,229, 7,018,523,7,267,939, U.S. patent application Ser. Nos. 09/096,593 and 60/980,733,and U.S. Provisional Application No. 61/087,102, filed on Aug. 7, 2008,all are herein incorporated in their entireties for all purposes.

Applications

The systems of the invention find use in the detection of a variety oftarget analytes, as outlined herein. In some embodiments, “sandwich”type assays are used, as are generally depicted in FIGS. 11-13. In otherembodiments, for example for the detection of particular metabolitessuch as ATP and NADH, other formats are used.

In some embodiments, for example in “sandwich” type formats, the targetanalyte, contained within a test sample, is added to the electrode withthe PSM-SIM-EAM mixture, a capture binding ligand, and optionally a SAM.This addition is followed by an optional washing step and the additionof the soluble binding ligand, although as will be appreciated by thosein the art, these additions can be done simultaneously or the solutionbinding ligand can be added to the sample containing the target analyteprior to addition to the chip. The surface is again optionally washed,and the substrate for the peroxide sensitive moiety, e.g. glucose, isadded under conditions that if present, peroxide is generated and theSIM is cleaved.

In some embodiments, for example when there is a specificperoxidase-generating enzyme that uses a metabolite of interest (e.g. atarget analyte or target metabolite), as depicted in FIGS. 12 and 13,the system takes on different configurations. For example, in FIG. 12,the electrode, generally with a SAM, contains at least two species: (1)an EAM with a self-immolative moiety, depicted in FIG. 12 as aferrocene-based derivative (although as described herein and as will beappreciated by those in the art, other EAMs can be used); and (2) anattached peroxidase-generating enzyme, in this case, as depicted, NADHoxidase. As outlined herein, the peroxidase-generating enzyme can beattached to the electrode surface in a number of different ways. Theattachment can be “direct” when the enzyme is attached to the terminusof a monolayer forming species, as is generally outlined inPCT/US2008/080379, using a coupling chemistry. Alternatively, and asdepicted in FIG. 12, the enzyme can be attached using a number of“sandwich” type attachments, for example using a monolayer species witha biotin attached, a streptavidin and a biotin-labeled enzyme.

In some embodiments, these two species can be attached as a singlemoiety to the electrode surface, for example as generally depicted inStructure 7 of U.S. Pat. No. 7,595,153, hereby incorporated by referencein its entirety and specifically for the schematics of attachmentconfigurations.

The ATP sensor can be based on a SAM-modified gold electrode thatcontains peroxide-reactive electroactive molecules (EAMs). The initialapparent formal potential of EAMs in the monolayer characterizes thesensor “off” state. Upon exposure to an ATP-containing sample matrix,hydrogen peroxide production triggers an irreversible eliminationreaction in the immobilized EAMs resulting in a signal “on” change inapparent formal potential of ferrocene groups that is detectedelectrochemically. This enzyme-triggered redox altering chemicalelimination (E-TRACE) reaction for a representative class of ferroceneEAMs is shown in FIG. 11. (see e.g. U.S. patent application Ser. No.12/853,204). In some embodiments, metabolites or other target analytesof interest can be detected on the basis of an event, such as anenzymatic event, occurring as the result of the presence of themetabolite or target analyte. For example, ATP can be detected inseveral ways. In one embodiment, an enzymatic event that relies on thepresence of ATP results in some sort of “switch” that then allowsdetection.

ATP is a multifunctional nucleotide that serves as the energy source formany biological processes in living organisms. Thus, strategies for thedetection of ATP have been a central focus for researchers in the fieldsof clinical diagnostics, microbiology, environmental analyses, and foodquality control. To date, numerous ATP sensors have emerged based oncolorometric, fluorometric, or electrochemical detection methods. Inparticular, sensors that employ ATP-dependent enzymatic reactions (i.ethose catalyzed by firefly luciferase, (Kadidate et al., Anal. Chem.2006 78:337-342 and DNA ligase, Wang et al., Biosens. Bioelectron. 201025:2101-2106, both of which are expressly incorporated by reference intheir entirety) to generate an output signal are attractive due to theirinherent sensitivity and specificity. In one embodiment, ATPelectrochemically is detected by exploiting the ATP-dependentbiotinylation reaction catalyzed by E. Coli biotin ligase (BirA) at aSAM-modified gold electrode. In another embodiment, ATP iselectrochemically detected by exploiting ATP-dependent enzymaticreactions with a SAM-modified gold electrode.

Among the class of enzymes that catalyze the post-translationalmodification of proteins with biotin, BirA has been shown tosite-specifically biotinylate lysine in the 15-amino acid acceptorpeptide (AP) sequence (GLNDIFEAQKIEWHE) with kinetics similar to thoseof the natural protein substrate (see Beckett et al, Protein Sci. 19998:921-929, expressly incorporated herein in its entirety). Thisenzymatic biotinylation reaction proceeds via two steps:

-   -   1.) biotin+ATP→biotinyl-5′-AMP+PPi    -   2.) biotinyl-5′-AMP+AP→biotin-AP+AMP

First, BirA catalyzes the formation of biotinyl-5′-AMP from biotin andATP. Subsequently, the target lysine residue on the dockedpeptide/protein substrate is biotinylated through nucleophilic attack onthis activated biotin intermediate (Cronan Cell 1989 58:427-429,expressly incorporated herein by reference in its entirety). Forexample, as depicted in FIG. 12, the electrode is formed with anattached acceptor peptide (AP) that serves as the substrate for thebiotin ligase enzyme, as well as an EAM with a self-immolative moiety. Asample containing ATP is added to the electrode in the presence of thebiotinylating enzyme and biotin, such that in the presence of ATP,biotin is added to the surface. A streptavidin-oxidase enzyme conjugateis then added, after optionally washing away the non-bound substrates,and then glucose is added, such that the peroxide generating enzymeresults in the destruction of the self-immolative linker and a change inthe redox potential (E⁰) of the ferrocene moiety.

In another embodiment, ATP is electrochemically exploited. In someembodiments, ATP can be electrochemically detected by exploiting theATP-dependent enzymatic reactions with a SAM-modified gold electrode byusing the activities of glycerol kinase (GK) and glycerol-3-phosphateoxidase (G3PO) if the co-substrate glycerol is present in the reactionmixture (see Murphy, L. J. et al. Anal. Chem. 1994, 66, 4345-4353; andLlaudet, E. et al., Anal. Chem. 2005, 77, 3267-3273). The enzymaticsteps proceed as follows:

-   -   1.) glycerol+ATP+GK→glycerol-3-phosphate+ADP    -   2.) glycerol-3-phosphate+G3PO+O₂→glycerone phosphate+H₂O₂

GK can catalyze the formation of glycerol-3-phosphate from glycerol andATP. Subsequently, G3PO oxidizes glycerol-3-phosphate to yield glyceronephosphate and hydrogen peroxide. Thus, the amount of peroxide producedis directly linked to the amount of ATP in the sample if glycerol is inexcess. An approach toward the electrochemical detection of peroxideproduced from this enzymatic reaction sequence is shown in FIG. 14.

Therein, each of the enzymes, such as G3PO and GK may be immobilized toa surface by chemical bonding (optionally via a linking group describedherein), may be physiochemically adsorbed with to a surface (e.g., viahydrophoblic interactions), or may be a homogeneous part of the solutionin contact with the electrode. The surface to which the enzymes areimmobilized or adsorbed may be a surface of the electrode, or a surfaceof any enclosing body (e.g., a reaction cell) which contains theelectrode and is in fluidly connected to the electrode via the solutionthat in contact with the electrode.

In certain embodiments, each of the enzymes are immobilized to theelectrode surface. In certain other embodiments, an oxidase isimmobilized to the substrate surface and any other enzyme may beimmobilized or adsorbed to the surface of the electrode, or ahomogeneous part of the solution in contact with the electrode.

These conditions are generally physiological conditions. Generally aplurality of assay mixtures is run in parallel with differentconcentrations to obtain a differential response to the variousconcentrations. Typically, one of these concentrations serves as anegative control, i.e., at zero concentration or below the level ofdetection. In addition, any variety of other reagents may be included inthe screening assay. These include reagents like salts, neutralproteins, e.g. albumin, detergents, etc which may be used to facilitateoptimal binding and/or reduce non-specific or background interactions.Also reagents that otherwise improve the efficiency of the assay, suchas protease inhibitors, nuclease inhibitors, anti-microbial agents,etc., may be used. The mixture of components may be added in any orderthat provides for the requisite binding.

The generation of peroxidase results in the loss of the PGM-SIMcomponent of the complex, resulting in a change in the E⁰ of the EAM. Insome embodiments, the E⁰ of the EAM changes by at about 20 mV, 30 mV, 40mV, 50 mV, 75 mV, 80 mV, 90 mV to 100 mV, some embodiments resulting inchanges of 200, 300 or 500 mV being achieved. In some embodiments, thechange in the E⁰ of the EAM is a decrease. In some embodiments, thechange in the E⁰ of the EAM is an increase.

The determination of solvent reorganization energy will be done as isappreciated by those in the art. Briefly, as outlined in Marcus theory,the electron transfer rates (kET) are determined at a number ofdifferent driving forces (or free energy, ΔG°); the point at which therate equals the free energy is the λ. This may be treated in most casesas the equivalent of the solvent reorganization energy; see Gray et al.Ann. Rev. Biochem. 65:537 (1996), hereby incorporated by reference.

The solvent inhibited redox active molecule, indicating the presence ofa target analyte, is detected by initiating electron transfer anddetecting a signal characteristic of electron transfer between thesolvent inhibited redox active molecule and the electrode.

Electron transfer is generally initiated electronically, with voltagebeing preferred. A potential is applied to a sample containing modifiednucleic acid probes. Precise control and variations in the appliedpotential can be via a potentiostat and either a three electrode system(one reference, one sample and one counter electrode) or a two electrodesystem (one sample and one counter electrode). This allows matching ofapplied potential to peak electron transfer potential of the systemwhich depends in part on the choice of redox active molecules and inpart on the conductive oligomer used.

Preferably, initiation and detection is chosen to maximize the relativedifference between the solvent reorganization energies of the solventaccessible and solvent inhibited redox active molecules.

Detection

Electron transfer between the redox active molecule and the electrodecan be detected in a variety of ways, with electronic detection,including, but not limited to, amperommetry, voltammetry, capacitanceand impedance being preferred. These methods include time or frequencydependent methods based on AC or DC currents, pulsed methods, lock intechniques, and filtering (high pass, low pass, band pass). In someembodiments, all that is required is electron transfer detection; inothers, the rate of electron transfer may be determined.

In some embodiments, electronic detection is used, includingamperommetry, voltammetry, capacitance, and impedance. Suitabletechniques include, but are not limited to, electrogravimetry;coulometry (including controlled potential coulometry and constantcurrent coulometry); voltametry (cyclic voltametry, pulse voltametry(normal pulse voltametry, square wave voltametry, differential pulsevoltametry, Osteryoung square wave voltametry, and coulostatic pulsetechniques); stripping analysis (aniodic stripping analysis, cathiodicstripping analysis, square wave stripping voltammetry); conductancemeasurements (electrolytic conductance, direct analysis); time dependentelectrochemical analyses (chronoamperometry, chronopotentiometry, cyclicchronopotentiometry and amperometry, AC polography, chronogalvametry,and chronocoulometry); AC impedance measurement; capacitancemeasurement; AC voltametry, and photoelectrochemistry.

In certain embodiments, amperommetry is used for electronic detection.The electrical current at the electrode can be determined in thepresence of and the absence of a target analyte, intermediary enzyme,co-factor, oxidase (e.g., peroxide generating enzyme), or other enzymesubstrate, to determine the presence and/or concentration of a targetanalyte. In each measurement, the current can be measured at the same ordifferent electrical potentials. For example, the electrical current canbe measured at a first E⁰ and at a second E⁰ after addition of a targetanalyte, intermediary enzyme, co-factor, oxidase (e.g., peroxidegenerating enzyme), or other enzyme substrate.

In some embodiments, monitoring electron transfer is via amperometricdetection. This method of detection involves applying a potential (ascompared to a separate reference electrode) between the electrodecontaining the compositions of the invention and an auxiliary (counter)electrode in the test sample. Electron transfer of differingefficiencies is induced in samples in the presence or absence of targetanalyte.

The device for measuring electron transfer amperometrically involvessensitive current detection and includes a means of controlling thevoltage potential, usually a potentiostat. This voltage is optimizedwith reference to the potential of the redox active molecule.

In some embodiments, alternative electron detection modes are utilized.For example, potentiometric (or voltammetric) measurements involve nonfaradaic (no net current flow) processes and are utilized traditionallyin pH and other ion detectors. Similar sensors are used to monitorelectron transfer between the redox active molecules and the electrode.In addition, other properties of insulators (such as resistance) and ofconductors (such as conductivity, impedance and capicitance) could beused to monitor electron transfer between the redox active molecules andthe electrode. Finally, any system that generates a current (such aselectron transfer) also generates a small magnetic field, which may bemonitored in some embodiments.

In some embodiments, the system may be calibrated to determine theamount of solvent accessible redox active molecules on an electrode byrunning the system in organic solvent prior to the addition of target.This is quite significant to serve as an internal control of the sensoror system. This allows a preliminary measurement, prior to the additionof target, on the same molecules that will be used for detection, ratherthan rely on a similar but different control system. Thus, the actualmolecules that will be used for the detection can be quantified prior toany experiment. Running the system in the absence of water, i.e. inorganic solvent such as acetonitrile, will exclude the water andsubstantially negate any solvent reorganization effects. This will allowa quantification of the actual number of molecules that are on thesurface of the electrode. The sample can then be added, an output signaldetermined, and the ratio of bound/unbound molecules determined. This isa significant advantage over prior methods.

It should be understood that one benefit of the fast rates of electrontransfer observed in the compositions of the invention is that timeresolution can greatly enhance the signal to noise results of monitorsbased on electronic current. The fast rates of electron transfer of thepresent invention result both in high signals and stereotyped delaysbetween electron transfer initiation and completion. By amplifyingsignals of particular delays, such as through the use of pulsedinitiation of electron transfer and “lock in” amplifiers of detection,orders of magnitude improvements in signal to noise may be achieved.

In some embodiments, electron transfer is initiated and detected usingdirect current (DC) techniques. As noted above, the E⁰ of the redoxactive molecule can shift as a result of the change in the solventreorganization energy upon target analyte binding. Thus, measurementstaken at the E⁰ of the solvent accessible redox active molecule and atthe E⁰ of the solvent inhibited molecule will allow the detection of theanalyte. As will be appreciated by those in the art, a number ofsuitable methods may be used to detect the electron transfer.

In some embodiments, electron transfer is initiated using alternatingcurrent (AC) methods. A first input electrical signal is applied to thesystem, preferably via at least the sample electrode (containing thecomplexes of the invention) and the counter electrode, to initiateelectron transfer between the electrode and the second electron transfermoiety. Three electrode systems may also be used, with the voltageapplied to the reference and working electrodes. In this embodiment, thefirst input signal comprises at least an AC component. The AC componentmay be of variable amplitude and frequency. Generally, for use in thepresent methods, the AC amplitude ranges from about 1 mV to about 1.1 V,with from about 10 mV to about 800 mV being preferred, and from about 10mV to about 500 mV being especially preferred. The AC frequency rangesfrom about 0.01 Hz to about 10 MHz, with from about 1 Hz to about 1 MHzbeing preferred, and from about 1 Hz to about 100 kHz being especiallypreferred

In some embodiments, the first input signal comprises a DC component andan AC component. That is, a DC offset voltage between the sample andcounter electrodes is swept through the electrochemical potential of thesecond electron transfer moiety. The sweep is used to identify the DCvoltage at which the maximum response of the system is seen. This isgenerally at or about the electrochemical potential of the redox activemolecule. Once this voltage is determined, either a sweep or one or moreuniform DC offset voltages may be used. DC offset voltages of from about1 V to about +1.1 V are preferred, with from about 500 mV to about +800mV being especially preferred, and from about 300 mV to about 500 mVbeing particularly preferred. On top of the DC offset voltage, an ACsignal component of variable amplitude and frequency is applied. If theredox active molecule has a low enough solvent reorganization energy torespond to the AC perturbation, an AC current will be produced due toelectron transfer between the electrode and the redox active molecule.

In some embodiments, the AC amplitude is varied. Without being bound bytheory, it appears that increasing the amplitude increases the drivingforce. Thus, higher amplitudes, which result in higher overpotentialsgive faster rates of electron transfer. Thus, generally, the same systemgives an improved response (i.e. higher output signals) at any singlefrequency through the use of higher overpotentials at that frequency.Thus, the amplitude may be increased at high frequencies to increase therate of electron transfer through the system, resulting in greatersensitivity. In addition, as noted above, it may be possible todistinguish between solvent accessible and solvent inhibited redoxactive molecules on the basis of the rate of electron transfer, which inturn can be used either to distinguish the two on the basis of frequencyor overpotential.

In some embodiments, measurements of the system are taken at least twoseparate amplitudes or overpotentials, with measurements at a pluralityof amplitudes being preferred. As noted above, changes in response as aresult of changes in amplitude may form the basis of identification,calibration and quantification of the system.

In some embodiments, the AC frequency is varied. At differentfrequencies, different molecules respond in different ways. As will beappreciated by those in the art, increasing the frequency generallyincreases the output current. However, when the frequency is greaterthan the rate at which electrons may travel between the electrode andthe redox active molecules, higher frequencies result in a loss ordecrease of output signal. At some point, the frequency will be greaterthan the rate of electron transfer through even solvent inhibited redoxactive molecules, and then the output signal will also drop.

In addition, the use of AC techniques allows the significant reductionof background signals at any single frequency due to entities other thanthe covalently attached nucleic acids, i.e. “locking out” or “filtering”unwanted signals. That is, the frequency response of a charge carrier orredox active molecule in solution will be limited by its diffusioncoefficient. Accordingly, at high frequencies, a charge carrier may notdiffuse rapidly enough to transfer its charge to the electrode, and/orthe charge transfer kinetics may not be fast enough. This isparticularly significant in embodiments that do not utilize apassivation layer monolayer or have partial or insufficient monolayers,i.e. where the solvent is accessible to the electrode. As outlinedabove, in DC techniques, the presence of “holes” where the electrode isaccessible to the solvent can result in solvent charge carriers “shortcircuiting” the system. However, using the present AC techniques, one ormore frequencies can be chosen that prevent a frequency response of oneor more charge carriers in solution, whether or not a monolayer ispresent. This is particularly significant since many biological fluidssuch as blood contain significant amounts of redox active moleculeswhich can interfere with amperometric detection methods.

In some embodiments, measurements of the system are taken at least twoseparate frequencies, with measurements at a plurality of frequenciesbeing preferred. A plurality of frequencies includes a scan. In apreferred embodiment, the frequency response is determined at least two,preferably at least about five, and more preferably at least about tenfrequencies.

Signal Processing

After transmitting the input signal to initiate electron transfer, anoutput signal is received or detected. The presence and magnitude of theoutput signal will depend on the overpotential/amplitude of the inputsignal; the frequency of the input AC signal; the composition of theintervening medium, i.e. the impedance, between the electron transfermoieties; the DC offset; the environment of the system; and the solvent.At a given input signal, the presence and magnitude of the output signalwill depend in general on the solvent reorganization energy required tobring about a change in the oxidation state of the metal ion. Thus, upontransmitting the input signal, comprising an AC component and a DCoffset, electrons are transferred between the electrode and the redoxactive molecule, when the solvent reorganization energy is low enough,the frequency is in range, and the amplitude is sufficient, resulting inan output signal.

In some embodiments, the output signal comprises an AC current. Asoutlined above, the magnitude of the output current will depend on anumber of parameters. By varying these parameters, the system may beoptimized in a number of ways.

In general, AC currents generated in the present invention range fromabout 1 femptoamp (fA) to about 1 milliamp (mA), with currents fromabout 50 fA to about 100 μA being preferred, and from about 1 pA toabout 1 μA being especially preferred.

Apparatus

The present invention further provides apparatus for the detection ofanalytes using AC detection methods. The apparatus includes a testchamber which has at least a first measuring or sample electrode, and asecond measuring or counter electrode. Three electrode systems are alsouseful. The first and second measuring electrodes are in contact with atest sample receiving region, such that in the presence of a liquid testsample, the two electrodes may be in electrical contact.

In yet another embodiment, the first measuring electrode comprises aredox active complex, covalently attached via a spacer, and preferablyvia a conductive oligomer, such as are described herein. Alternatively,the first measuring electrode comprises covalently attached redox activemolecules and binding ligands.

The apparatus further comprises a voltage source electrically connectedto the test chamber; that is, to the measuring electrodes. Preferably,the voltage source is capable of delivering AC and DC voltages, ifneeded.

In an embodiment, the apparatus further comprises a processor capable ofcomparing the input signal and the output signal. The processor iscoupled to the electrodes and configured to receive an output signal,and thus detect the presence of the target analyte.

EXAMPLES Example 1 Preparation of Compounds 1-5 and ElectrochemicalEvaluation

General Methods and Materials. Unless otherwise noted, all syntheticmanipulations were performed under a dry argon atmosphere using standardSchlenk techniques. For reaction media, solvents were dried over neutralalumina via the Dow-Grubbs solvent system acquired from Glass Contours(Laguna Beach, Calif.). These solvents were deoxygenated with argonprior to use. Reactions were monitored by TLC using EMD pre-coatedaluminum plates (silica gel 60, F₂₅₄, EMD Chemicals, Inc., Gibbstown,N.J.). Spots were visualized by one of the following methods: iodinevapor, exposure to UV light, or staining with phosphomolybdic acidfollowed by heating. Flash chromatography was carried out on silica(silica gel 60 particle size: 40-63 μm; Sorbent Technologies, Atlanta,Ga.) under a positive pressure of laboratory air. ¹H NMR andproton-decoupled ¹³C NMR spectra were recorded on a Bruker Avance IIIspectrometer (499.37 MHz for ¹H, 125.58 MHz for ¹³C) and were processedwith Bruker TOPSPIN 2.1 software. High-resolution mass spectrometry(HRMS) was obtained using an Agilent 6210 time-of-flight (TOF) LC/MSinstrument using electrospray ionization (ESI) or atmospheric pressurephotoionization (APPI) methods.

Chloroform-d₁ was purchased from Cambridge Isotope Laboratories.Compound 2 and p-pinacolborate benzyl alcohol were synthesized asdescribed previously (Bertin, P. A.; Meade, T. J. Tetrahedron Lett.2009, 50, 5409-5412; Sella, E.; Shabat, D. Chem. Commun. 2008,5701-5703, both of which are expressly incorporated by reference). Allother reagents were purchased from commercial sources and used withoutfurther purification unless otherwise noted.

Compound 3. To a solution of compound 2 (0.500 g, 1.2 mmol) andtriethylamine (0.25 mL, 1.8 mmol) in tetrahydrofuran (15 mL) was addeddiphenylphosphoryl azide (DPPA) (0.285 mL, 1.32 mmol). The reaction wasstirred at room temperature for 1.5 hours and concentrated under reducedpressure. The crude residue was purified by column chromatography(methanol:ethyl acetate:dichloromethane 0.5:1.5:8) to yield the titlecompound as a red/orange solid (0.460 g, 1.04 mmol, 87%). ¹H NMR,¹³C{¹H} NMR, and HRMS were consistent with the title compound.

Compound 4. A solution of compound 3 (0.460 g, 1.04 mmol) in toluene (20mL) was vigorously degassed with Ar and heated to 100° C. for 1.5 hours,p-pinacolborate benzyl alcohol (0.268 g, 1.14 mmol) anddibutyltin-dilaurate (DBTL) (0.018 mL, 0.03 mmol) were added and thereaction maintained at 100° C. for an additional 2 hours. The reactionwas concentrated under reduced pressure and the crude residue purifiedby column chromatography (diethyl ether:ethyl acetate:dichloromethane,1:2:2) to yield the title compound as a pale orange solid (0.480 g,0.741 mmol, 71%). ¹H NMR, ¹³C{¹H} NMR, and HRMS were consistent with thetitle compound.

Compound 5. A solution of compound 4 (0.135 g, 0.209 mmol) indichloromethane (5 mL) was cooled in an ice bath. Trifluoroaceticacid:dichloromethane (1:1 v/v, 5 mL) was added dropwise over 5 min.After 15 min, the ice bath was removed and the reaction warmed to roomtemperature. After 45 min, the volatiles were removed in vacuo to yieldthe trifluoroacetate salt of the title compound as a brown/orange solid(quantitative). ¹H NMR, ¹³C{¹H} NMR, and HRMS were consistent with thetitle compound.

Compound 1. To a solution of 11-mercaptoundecanoic acid (0.045 g, 0.206mmol) and HATU (0.078 g, 0.206 mmol) indichloromethane:N,N-dimethylformamide (1:1 v/v, 5 mL) was added compound5 (0.105 g, 0.159 mmol) and diisopropylethylamine (0.083 mL, 0.477mmol). The reaction was stirred at room temperature for 2 hours. Thereaction mixture was diluted into ethyl acetate (150 mL) and washed withbrine (3×50 mL). The organic phase was dried over Na₂SO₄, filtered, andconcentrated to crude residue that was purified by column chromatography(methanol:ethyl acetate:dichloromethane, 0.5:1.5:8) to yield the titlecompound as a yellow solid (0.035 g, 0.047 mmol, 30%). ¹H NMR, ¹³C{¹H}NMR, and HRMS were consistent with the title compound.

Electrochemistry. Cyclic voltammetry was carried out with a CHI model660A electrochemical analyzer (CHI Instruments Inc.) in THF with 0.15 Mn-Bu₄NCIO₄ supporting electrolyte (0.5 mL) using a three electrodesystem of SAM-modified gold as the working electrode, a Ag/AgCl wirereference electrode, and a platinum wire counter electrode(Bioanalytical Systems). Model compound (green) was prepared by treatingcompound 4 with hydrogen peroxide. The results are shown in FIG. 3.

Example 2 Change in E⁰ as a Result of the Presence of H₂O:H₂O, Study onPB25_(—)49 Diluted with C6 Diluent; 5-Minute and 10-Minute Incubationsin Na₂CO₃ buffer (pH 10.1) A. Purpose

The goal of this study was to test the effect of 5-minute and 10-minuteH₂O₂ incubation times on a diluted SAM of EAM 1 (PB25_(—)49) washed atpH 10.1 and incubated at pH 8.5. H₂O₂ would decompose the ferrocene onthe EAM into a new derivative which would show up at a new potential.

B. Materials

BATCH #/ Stock/ MATERIALS Name MW Final C Solvent NOTES 1. EAM for SAM:PB25_49 MW = 747.57  0.1 mM 0.5 mg/0.5 mL Stock: 0.5 mg ethanol 2.Diluent solutions (C₆S)₂ MW = 234.47  0.5 mM — Stock C = 9.13× (4.56 mMstock) for SAM (HO—C₆S)₂ MW = 266.47  0.5 mM — Stock C = 7.51× (3.75 mMstock) 3. Electrode testing 1M LiClO₄ MW = 106.39 1M 10.6 g/1 L H₂OAqueous solution solution: 1× 4. Hydrogen peroxide Hydrogen MW = 34.011M 57 μL/ 943 μL Made fresh peroxide H₂O (50.4%) 5. Buffer Na₂CO₃ MW =105.99  100 mM 0.53 g/50 mL pH 10.1 H₂O 6. Washing buffers ethanol, — —— — nanopure water, Na₂CO₃, 1M LiClO₄ 7. Electrodes: Reference CounterWorking Wash and store — electrode electrode Electrode Quasi 1 Pt WireAu Chip Rinse before 13 green chips reference d = 0.25 and after each(1M LiClO₄) um use (C₆S)₂: dihexyl disulfide; (HO—C₆S)₂:bis(6-hydroxyhexyl) disulfide; Note: Green chips were printed circuitboard gold electrode arrays obtained from Osmetech; Nanopure water, asused herein means18 megaohm water from a Millipore (Billerica, MA, USA)purification system.

EAM 1 Structure:

C. Procedure Day 1: Wash and Assemble Chips

-   -   13 (12 for assay+1 for internal reference testing) green chips        were washed as follows:    -   Placed chips in a glass jar with inserts    -   Sonicated in 0.2% TWEEN™ 20 solution for 5 minutes    -   Rinsed with nanopure water and ethanol, then dried with Argon    -   Cleaned in a Plasma Cleaner for 10 minutes    -   Rinsed with ethanol and dried with Argon    -   Metal bases, gaskets, and PDMS stamps were washed as follows:    -   Hand washed with hand soap    -   Rinsed with ethanol and air dried    -   Chips were assembled by placing the chip on the double sided        tape on center of the metal base, PDMS stamp on the chip, gasket        on the PDMS stamp, all clamped together with binder clips.

Prepare Experimental Stocks

EAM stock was prepared by combining the following into the EAM aliquot:

# Etha- Final Stocks EAM Aliquots Amount MW nol THF Conc. A PB25_49 10.5 mg 747.57 500 none 1.34 ea μL mM

Prepare SAM Solutions

SAM Solution was prepared by combining the following into separate glassvials:

Tot AMT AMT AMT Ethanol Vol. [EAM] μL D1 [D1] μL D2 [D2] μL μL μL 1.34mM 493 (C₆S)₂ 4.56 mM 723 (HOC₆S)₂ 3.75 mM 879 4505 6600

SAM Incubation

To chips 1-12, 500 μL of above prepared SAM solution was added, followedby overnight incubation.

All chips were placed in plastic containers containing ethanol, sealedwith parafilm (to avoid ethanol evaporation and drying of chips). Thesetup was covered with Aluminum foil.

Day 2: Internal Reference Measurements:

A 1 mM solution of 1 1′-ferrocene dimethanol was prepared in 1M LiClO₄solution. 1.3 mg of 1 1′-ferrocene dimethanol were combined with 5 mL 1MLiClO₄. MW 1 1′-ferrocene dimethanol=246.09

500 μL of 1 mM 1 1′-ferrocene dimethanol solution was added to a cleanchip. Quasi 1 reference and platinum counter electrodes were added tothe system and CVs were recorded.

Initial Testing to Check for Proper SAM Formation on Chips:

After overnight incubation, the chips were removed from the incubationcontainer. The SAM deposition solution was collected in a vial and driedto obtain recycled EAM for future use.

After overnight incubation, chips 1 through 12 were washed by followingthe steps shown below:

Ethanol 8 times Nanopure water 4 times Testing buffer, 1M LiClO₄ 2 times500 μL of 1M LiClO₄ was added to chips 1, 3, 5, 6, 7, 9, 11, and 12, andthen chip was plugged in the switchbox.

Reference and counter electrodes were added to the EC system. The whitealligator clip from the CHI 650C was connected to the referenceelectrode (Quasi 1), green clip to the working electrode and the redclip to the counter electrode (Platinum wire, flamed in advance, rinsedwith ethanol and water).

The CHI 650C system was used to test all chips. For each test, six fileswere used: 10000 mV/s, 100 mV/s, 10000 mV/s long, multi CV (20 cycles)and ACV (forward and backward).

The multiplexer was used for testing all chips in this experiment.

After initial testing, chips 1 through 5 and 7 through 11 were washed asfollows:

Nanopure water 8 times 100 mM Na₂CO₃ (pH 10.1) 2 timesChips 6 and 12 were washed as follows:

Nanopure water 8 times 100 mM NaHCO₃ (pH 8.5) 2 times

Preparation of Different Concentrations of Hydrogen Peroxide:

Different concentrations of H₂O₂ solution were made in 100 mM Na₂CO₃buffer (pH 10.1) immediately before use. Original stock of H₂O₂ was at1M which was made by combining 57 μL of 50% H₂O₂ with 943 μL of nanopurewater. This stock was left in the 4° C. fridge overnight to allow formuta-rotation. From there on the dilutions were made as shown below:

Final Ratio Amount of Amount concentration (previous to previous ofTotal of H₂O₂ final concentration buffer volume (mM) concentration) ofH₂O₂ (μL) (μL) (μL) 1 1:1000 2 1998 2000 0.1 1:10  200 1800 2000 0.011:10  200 1800 2000 0.001 1:10  200 1800 2000 0 — 0 2000 2000

Addition of Different Concentrations of H₂O₂ to the Chips and Testing:

The hydrogen peroxide solutions made were vortexed well.

500 μL of the respective hydrogen peroxide solutions was added to eachchip (1-12) and the solution was mixed thoroughly. The incubation wascarried out at room temperature for 5 minutes, while mixing the solutionin between with pipette tips at 4:30, 2:30, and 0:30 times, and for 10minutes, while mixing the solution in between with pipette tips at 7:30,5:00, and 2:30 times.

After the respective H₂O₂ incubations, the chips were washed as follows:

Nanopure water 8 times 100 mM Na₂CO₃ (pH 10.1) or 2 times 100 mM NaHCO₃(pH 8.5)Each well was incubated with 500 μL of their respective buffers for 5minutes. After the chips were incubated with buffer, the chips werewashed as follows:

Nanopure water 8 times 1M LiClO₄ 2 times

The switchbox was used for testing all chips as shown in steps VI d, eand f.

After testing, the chips were washed, cleaned with ethanol and water andthen disassembled.

Experiment Outline

Chip Chip Name 1 #1_2_H2O2_0uM_5min_pH10pt1 2 #2_2_H2O2_1uM_5min_pH10pt13 #3_2_H2O2_10uM_5min_pH10pt1 4 #4_2_H2O2_100uM_5min_pH10pt1 5#5_2_H2O2_1mM_5min_pH10pt1 6 #6_2_H2O2_0uM_5min_pH8pt5 7#7_2_H2O2_0uM_10min_pH10pt1 8 #8_2_H2O2_1uM_10min_pH10pt1 9#9_2_H2O2_10uM_10min_pH10pt1 10 #10_2_H2O2_100uM_10min_pH10pt1 11#11_2_H2O2_1mM_10min_pH10pt1 12 #11_2_H2O2_0uM_10min_pH8pt5 13#11_3_post-H2O2_FcMe2

Example 3 H₂O Study on PB25_(—)49 Diluted with C₆ Diluents; 5-Minute and10-Minute Incubations in NaHCO₃ buffer (pH 8.5) with 100 uM Glucoseoxidase (GO_(x)) Purpose

The goal of this study was to test the effect of 5-minute and 10-minuteglucose incubation times on a diluted SAM of PB25_(—)49 washed at pH10.1 and incubated at pH 8.5. Glucose oxidase was added at 100 μM tothese chips to produce H₂O₂ that would decompose the ferrocene on theEAM into a new derivative which would show up at a new potential.

B. Materials

MATERIALS BATCH #/Name MW Final C Stock/Solvent NOTES 1. EAM for SAM:PB25_49 MW = 747.57  0.1 mM 0.5 mg/0.5 mL Stock: 0.5 mg ethanol 2.Diluent solutions (C₆S)₂ MW = 234.47  0.5 mM - Stock C = 9.13× (4.56 forSAM mM stock) (HO—C₆S)₂ MW = 266.47  0.5 mM - Stock C = 7.51× (3.75 mMstock) 3. Electrode testing : 1M LiClO₄ MW = 106.39 1M 10.6 g/1 L H₂OAqueous solution solution 1× 4. Glucose Glucose MW = 180.1 1M 0.99 g/5mL water Muta-rotated overnight, monohydrate 4° C. 5. Glucose oxidaseGO_(x) MW = 160000  100 μM 12.8 mg/800 Made fresh on day of μLNaHCO₃buffer use 6. Buffer NaHCO₃ MW = 105.99  100 mM 4.2 g/500 mL pH 8.5 7.Washing buffers ethanol, nanopure — — — — water, NaHCO₃, Na₂CO₃, buffer,1M LiClO₄ 8. Electrodes: Reference Counter Working Wash and store —electrode electrode Electrode Quasi 1 reference Pt Wire Au Chip Rinsebefore and 11 green chips (1M LiClO₄) d = 0.25 after each use um

EAM structure is as shown:

C. Procedure Day 1: Wash and Assemble Chips

-   -   11 (10 for assay+1 for internal reference testing) green chips        were washed as follows:    -   Placed chips in a glass jar with inserts    -   Sonicated in 0.2% TWEEN™ 20 solution for 5 minutes    -   Rinsed with nanopure water and ethanol, then dried with Argon    -   Cleaned in a Plasma Cleaner for 10 minutes    -   Rinsed with ethanol and dried with Argon    -   Metal bases, gaskets, and PDMS stamps were washed as follows:    -   Hand washed with hand soap    -   Rinsed with ethanol and air dried    -   Chips were assembled by placing the chip on the double sided        tape on center of the metal base, PDMS stamp on the chip, gasket        on the PDMS stamp, all clamped together with binder clips.

Prepare Experimental Stocks

EAM stock was prepared by combining the following into the EAM aliquot:

# Etha- Final Stocks EAM Aliquots Amount MW nol THF Conc. A PB25_49 10.5 mg 747.57 500 none 1.34 ea μL mM

Prepare SAM Solutions

SAM Solution was prepared by combining the following into separate glassvials:

Tot AMT AMT AMT Ethanol Vol. [EAM] μL D1 [D1] μL D2 [D2] uL μL μL 1.34mM 411 (C₁₁S)₂ 2.67 mM 1030 (HOC₁₁S)₂ 2.46 mM 1119 2940 5500

SAM Incubation

To chips 1-10, 500 μL of above prepared SAM solution was added, followedby overnight incubation.

All chips were placed in plastic containers containing ethanol, sealedwith parafilm (to avoid ethanol evaporation and drying of chips). Thesetup was covered with aluminum foil.

Day 2: Internal Reference Measurements:

A 1 mM solution of 1 1′-ferrocene dimethanol was prepared in 1M LiClO₄solution. 1.3 mg of 1 1′-ferrocene dimethanol were combined with 5 mL 1MLiClO₄. MW 1 1′-ferrocene dimethanol=246.09

500 μL of 1 mM 1 1′-ferrocene dimethanol solution was added to a cleanchip. Quasi 1 reference and platinum counter electrodes were added tothe system and CVs were recorded.

Initial Testing to Check for Proper SAM Formation on Chips:

After overnight incubation, the chips were removed from the incubationcontainer. The SAM deposition solution was collected in a vial and driedto obtain recycled EAM for future use.

After overnight incubation, chips 1 through 10 were washed by followingthe steps shown below:

Ethanol 8 times Nanopure water 4 times Testing buffer, 1M LiClO₄ 2 times500 μL of 1M LiClO₄ was added to chips 1, 3, 5, 6, 8, and 10, and thenchip was plugged in the switchbox.

Reference and counter electrodes were added to the EC system. The whitealligator clip from the CHI 650C was connected to the referenceelectrode (Quasi 1), green clip to the working electrode and the redclip to the counter electrode (Platinum wire, flamed in advance, rinsedwith ethanol and water).

The CHI 650C system was used to test all chips. For each test, six fileswere used: 10000 mV/s, 100 mV/s, 10000 mV/s long, multi CV (20 cycles)and ACV (forward and backward).

The multiplexer was used for testing all chips in this experiment.

After initial testing, chips 1 through 10 were washed as follows:

Nanopure water 8 times 100 mM NaHCO₃ 2 times

Preparation of Different Concentrations of Glucose:

A 100 μM Stock of GO_(x) was made by combining 12.8 mg of GO_(x) with800 μL of NaHCO₃ buffer, immediately before use.

Different concentrations of glucose solution were made in 100 mM NaHCO₃buffer (pH 8.5) immediately before use. Original stock of glucose was at1M which was made by combining 0.99 g of Glucose with 5 mL nanopurewater. This stock was left in the 4° C. fridge overnight to allow formuta-rotation. From there on the dilutions were made as shown below:

Final Ratio Amount of concentration (previous to previous Amount Totalof Glucose final concentration of buffer volume (mM) concentration) ofGlucose (μL) (μL) (μL) 1 1:1000 2 1998 2000 0.1 1:10  200 1800 2000 0.011:10  200 1800 2000 0.001 1:10  200 1800 2000 0 — 0 2000 2000

Addition of Different Concentrations of Glucose to the Chips andTesting:

The glucose solutions made were vortexed well and 450 μL were added tothe respective chips.

50 μL of 100 uM Glucose oxidase was added to each chip (1-10) and thesolution was mixed thoroughly. The incubation was carried out at roomtemperature for 5 minutes, while mixing the solution in between withpipette tips at 4:30, 2:30, and 0:30 times, and for 10 minutes, whilemixing the solution in between with pipette tips at 7:30, 5:00, and 2:30times.

After the respective glucose incubations, the chips were washed asfollows:

Nanopure water 8 times Na₂CO₃, pH 10.1 2 timesEach well was incubated with 500 μL of Na₂CO₃, (pH 10.1) buffer for 5minutes.

After the chips were incubated with buffer, the chips were washed asfollows:

Nanopure water 8 times 1M LiClO₄ 2 timesThe switchbox was used for testing all chips as shown in steps VI d, eand f. After testing, the chips were washed, cleaned with ethanol andwater and then disassembled.

Experiment Outline

Chip Chip Name 1 #1_2_glucose_0 mM_5 min 2 #2_2_glucose_1 uM_5 min 3#3_2_glucose_10 uM_5 min 4 #4_2_glucose_100 uM_5 min 5 #5_2_glucose_1mM_5 min 6 #6_2_glucose_0 mM_10 min 7 #7_2_glucose_1 uM_10 min 8#8_2_glucose_10 uM_10 min 9 #9_2_glucose_100 uM_10 min 10#10_2_glucose_1 mM_10 min 11 #11_3_post-glucose_FcMe2

Study: Testing PB25_(—)49 on Green Chips with Troponin

A. Purpose

The goal of this study was to determine the electrochemistry of the EAMPB25_(—)49 on green chips after creating a troponin antibody sandwichand glucose addition resulting in H₂O₂ generation due to secondaryantibody GO_(x) labeling.

B. Materials

MATERIALS BATCH #/Name MW ES Conc Stock/Solvent NOTES 1. SAM EAM:PB25_49 747.57  0.1 mM 0.5 mg/ Prepared 0.5 mL Previously ethanol 2. SAM(OH—C₁₁—S)₂ 406.72  0.5 mM 1 mg/mL Prepared Diluent Previously PreparedII (C₁₁S)₂ 374.72  0.5 mM 5 mM Previously HS—C₁₆—COOH 288.49 0.001 mM0.5 mg 3. Incubation PBS — — — — Buffer 4. Testing 1M LiClO₄ 106.39 1M10.6 g/L Prepared solution: H₂O Previously 5. Washing ethanol, nanopure— — — buffers water, 1M LiClO₄, PBS 6. Electrodes: Reference CounterWorking Wash and store — Quasi 4 Pt Wire Au Chip Rinse before and 10Green (1M LiClO₄) d = 0.25 μm after each use

C. Procedure

Day 1:

Prepare SAM Solution

The following Experimental Stocks were prepared by combining the stockmaterial to the corresponding solvents and additives.

EAM: One 0.5 mg PB25_(—)49 aliquots

Add 500 μL ethanol

Bis(11-hydroxyundecyl) disulfide (HO—C₁₁—S)₂: Pre-made 1 mg/mL stock

Diundecyl disulfide, (C₁₁—S)₂: Pre-made 1 mg/mL stock

HS—C₁₆—COOH (16-mercaptohexadecanoic acid) Added 500 μL to 0.5 mgaliquot

The SAM solution was prepared by combining the following in a 20 mLglass vial:

PB25_(—)49: 411 μL of 1.34 mM ES (estimated stock concentration) forfinal concentration of 0.1 mM

(OH—C₁₁—S)₂: 1.118 mL of 2.46 mM ES for final concentration of 0.5 mM

(C₁₁—S)₂: 1.030 mL of 2.67 mM for final concentration of 0.5 mM

HS—C₁₆—COOH: 0.002 mL of 3.47 mM ES for a final concentration of 0.001mM

Ethanol (EtOH): 2.940 mL for a total volume of 5.5 mL

SAM Deposition

For all chips, the following procedure was performed to deposit the SAM:Chips were placed in slotted microscope-slide jar with exposed goldsurfaces facing inwardsPre-made 0.2% TWEEN™ 20 was added to the jar until the chips werecompletely submergedAfter sonicating, the chips were thoroughly rinsed with nanopure waterEach chip rinsed with ethanol and dried with argon gasThe chips were plasma cleaned for 10 minutes at the “low” plasma settingAfter plasma cleaning, the chips were again rinsed with ethanol anddried with argon Accessory parts (base, gasket, tub) cleaned byscrubbing with hand soap, rinsing with deionized water and nanopurewater, rinsing with ethanol, and air-dryingChips were assembled, then leak tested with ethanol to ensure the gasketwas producing a good seal500 μL of the deposition solution prepared above was added to the tub ineach chipChips were incubated overnight at in a sealed and covered glasscontainer

Day 2:

Initial Testing to Verify Proper SAM Formation

Following overnight incubation, chips removed from containers. The chipswere washed as follows:

2× Ethanol

6× Nanopure water

2×LiClO₄

500 μL testing solution (see table above) was added in each tubThe electrodes (see table above) were connected to the CHI system (thePt counter was cleaned with a propane torch and ethanol rinse prior touse)For all chips:Cyclic voltammetry was performed between ranges determined duringtesting with 10000 mV/s CV, 100 mV/s CV,

EDC, NHS Activation

The Chips were washed 4× with nanopure water before addition of anyfurther solutions.

Added 1000 μL of EDC to 1000 μL of NHS.

Added 200 μL of this mixed solution to 4 chipsIncubate for 30 minutes. NOTE: All incubations were done in emptypipette tip containers and covered with foil to minimize light exposure.Wash 4× with nanopure water after incubation

Streptavidin

Add 200 μL streptavidin solutionIncubated for 1 hour

Wash 4×PBS. 1×LiClO₄.

Chips 3-6 were tested as in 3.5.1NOTE: only the chips that were tested were washed with LiClO₄. All chipsthat were tested were washed again 4×PBS. This applies to all stepsbelow.

Chip Material 1 Gox- Biotin 2 Gox- Biotin 3 Tested after each step 4Tested after each step 5 Tested after each step 6 Tested after each step7 Tested only immediately before glucose 8 Tested only immediatelybefore glucose 9 Only tested after addition of glucose 10 Only testedafter addition of glucose

Ethanolamine Capping

200 μL ethanolamine was added to each chipIncubate for 15 minutes

Wash 4×PBS

BSA Blocking

Add 0.1% BSA (bovine serum albumin)Incubate 10 minutesWash 4×PBS (phosphate buffered saline), 1×LiClO₄Chips 3-6 were tested as in 3.5.1

GO_(x) and Primary Antibody Addition

Concentration of GO_(x)-biotin stock is 1 mg/mL so 4 μL of GO_(x)-biotinstock was added to 396 μL PBS.Added 200 μL of 10 μg/mL GO_(x)-biotin to Chip #1&2.Incubated for 1 hourStock of mAb 19C7-biotin is at 1.7 mg/mL so 8 μL was added to 792 μL ofPBS for a concentration of 17 μg/mLAdded 100 μL antibody-biotin to #3-10Both solutions were left to incubate for 45 minutes.Chips 3-6 were washed 4×PBS, 1×LiClO₄ and tested.

Troponin and Secondary Antibody Incubation

The troponin aliquot (1 mg/mL) was taken from the −20° C. freezer andlet thaw. 1 μL was added to 9 μL of PBS, yielding 100 μg/mL. NOTE: thisdilution was not done in the Urea/tris buffer.1 μL of the 100 μg/mL was added to 48 μL of PBSmAb16A11-Gox was removed from the fridge (1.7 mg/mL)1 μL of mAb16A11-Gox was also added to the PBS/troponin solution andvortex.This solution was incubated for 30 mins.

Gox-Biotin Testing

-   -   Chips #1, #2 were washed 4×PBS, 1×LiClO₄ and tested.    -   After testing they were washed 4×PBS.    -   A 2 mM Glucose solution was prepared by taking 20 μL of 1M to        9980 μL of NaHCO₃ pH 8.5.    -   500 μL of 2 mM glucose was added to both chips and incubated for        10 minutes.    -   #1,2 were then washed 4×PBS, 1×LiClO₄ and tested.    -   Chips were washed 4×PBS    -   Chips were incubated with 100 mM H₂O₂ for 2 minutes, then washed        and tested

Troponin and Secondary Antibody Addition

-   -   Chips 3-10 were washed 4×PBS.    -   The 50 μL incubation of troponin and mAb16A11-Gox was diluted up        to 1.2 mL in PBS. Solution was vortexed    -   150 μL of this solution was added to chips 3-10 and incubated        for 30 mins.

Troponin and Secondard Antibody Testing.

-   -   Chips 3-10 were washed 4×PBS.    -   Chips 5-8 were washed 1× LiClO₄ and tested.    -   Chips 5-8 were washed with 4×PBS    -   500 μL of 100 mM H₂O₂ was added to #6-7 for 2 minutes. After        incubation they were washed 4×PBS, 1×LiClO₄ and tested.    -   2 mM glucose was added to Chips 9, 10 and incubated for 20        minutes. After incubation they were washed 4×PBS, 1×LiClO₄ and        tested.

Monolayer Preparation. Gold-evaporated electrodes were cleaned with a 5minute sonication in 0.2% Tween 20 solution, washed with ethanol beforeundergoing 10 minutes of plasma ionization. The electrodes were thenwashed with ethanol before being exposed to the deposition solution. Thedeposition solution was composed of Compound 1 (0.1 mM), dihexyldisulfide (0.5 mM (C₆—S)₂) and bis(6-hydroxyhexyl) disulfide (0.5 mM(HO—C₆—S)₂) and 16-mercaptohexadecanoic acid (0.01 mM, MHA,HS—C₁₆—COOH). The deposition solution was incubated on the goldelectrodes overnight for ˜18 hours. The deposition solution was thenremoved and the electrodes were washed with ethanol followed by water.The MHA was activated for 30 minutes using a 1/1 volume/volume ofN-hydroxysuccinimide (NHS) (0.1 M) and1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (0.4 M). Followingthis activation the electrodes were washed with water and incubated 1hour with streptavidin (0.05 mg/mL) in 10 mM sodium acetate pH 5.7. Theelectrodes were washed with PBS between each step for the remainingsteps of the assay. Ethanolamine (0.1 mM NaHCO₃) was added to cap theunreacted MHA sites for 15 minutes. BSA (0.1 weight %) in PBS buffer wasadded for 10 minutes to reduce nonspecific binding. The primary antibody(mAb-19c7-biotin, HyTest) was added at a concentration of 17 μg/mL andincubated for 45 minutes. During this time, the secondary antibody (34μg/mL, mAb-16A11-GOx, Hytest) was incubated with Human cardiac troponinI (2 μg/mL). The secondary antibody-troponin complex was then added tothe electrodes at concentration of interest and incubated for 30minutes. At this point the full “sandwich” is built up on the MHA in themonolayer. Glucose (2 mM) was then incubated for 10 minutes, solutionwas removed, electrodes were washed with PBS and the cyclicvoltammograms were recorded in LiClO₄ (1 M).

Example 4 NADH Dose Response for Complete Lysis Buffer with Zinc Acetate

Purpose: The purpose of this experiment is to get dose response for NADHwithout amplification system for complete lysis buffer with zinc acetate

Materials and Methods

Protease Inhibitor Calbiochem 539134 and VWR 80053-85 Benzonase Novagen#70664

NADH oxidase (GTP, Aliquot: 0.4 U/50 μL, tube; =17.2 μg/tube; =0.344μg/μL

Buffer Used:

0.5×HEPES, pH 7.4 (29 mM Maltoside) 1:200 Benzonase

1:2000 Protease inhibitor

Phosphatase Inhibitor Cocktail (0.5×)

2.5 mM NaF

3 μM Sodium Orthovanadate (instead of 500 μM)500 μM Sodium Pyrophosphate decahydrate500 μM beta-Glycerolphosphate

2.5 mM Sodium Azide

100 μM zinc acetate

Desired Concentrations in Well/Tube

Solution Concentration NADH oxidase    5 ng/μL FAD   10 μM NADH    3 μM   1 μM  300 nM  100 nM   30 nM   10 nM    3 nM    1 nM  0.3 nM

Working Solutions

Working NADH oxidase: Take NADH oxidase aliquot 50 μL. Add 1.45 μL ofstock per 100 μL of total NADH reaction solution.

FAD: Make a 7.5 mM stock solution by dissolving 5.8 mg in 1000 μLnanopure water. Use 7.5 mM stock and make 1 mM stock by taking 13.3 μLof 7.5 mM stock and diluting upto 100 μL. Add 1 μL per 100 μL reaction.

ADHP: Add 220 μL to 100 μL stock of 16 mM ADHP stock to give 5 mM stock.Add 2.3 μL/70 μL in each well to get 160 μM conc.

HRP: Add 0.6 μL of 1 u/μL HRP to 999.4 μL water. Add 2.3 μL HRP/70 μLreaction to each well

NADH:

Final conc Water (10 μL in 100 μL NADH Conc Stock (mM) μL sample)  10 mM10 —  1 mM   3 mM 30 70 300 μM   1 mM 30 60 100 μM 300 μM 30 70  30 μM100 μM 30 60  10 μM  30 pM 30 70  3 μM  10 μM 30 60  1 μM   3 μM 30 70300 nM   1 μM 30 60 100 nM 300 nM 30 70  30 nM   0 mM — 60  0 Unknown —— —

Reaction: Composition Per Well

Complete lysis buffer with Take NADH inhibitor sample NADH cocktail and(1 mM-30 nM) FAD oxidase sample benzonase Buffer μL 1 mM 0.344 ug/μL 1HEPES buffer 90 μL 10 μL 1 μL 1.45 μL 2 HEPES buffer 90 μL 10 μL 1 μL1.45 μL 3 HEPES buffer 90 μL 10 μL 1 μL 1.45 μL 4 HEPES buffer 90 μL 10μL 1 μL 1.45 μL 5 HEPES buffer 90 μL 10 μL 1 μL 1.45 μL 6 HEPES buffer90 μL 10 μL 1 μL 1.45 μL 7 HEPES buffer 90 μL 10 μL 1 μL 1.45 μL 8 HEPESbuffer 90 μL 10 μL 1 μL 1.45 μL 9 HEPES buffer 90 μL 10 μL 1 μL 1.45 μL10 HEPES buffer 90 μL 10 μL 1 μL 1.45 μL 11 HEPES buffer 90 μL 10 μL 1μL 1.45 μL 12 HEPES buffer — 100 μL  1 μL 1.45 μL (cell lysate)

100 μL sample each.

1. Each component was added to a 96 well plate and incubated for 2hours.

2. After 2 hrs 30 μL from each reaction sample was taken and pH adjustedto pH 10 by adding 10 μL of 400 mM carbonate. 30 μL was then incubatedon e-chips.

3. 70 μL was taken and added to ADHP and HRP for optical readout

4. A Corning Polystyrene 96-well plate was used for this experiment. Theappropriate filters were inserted into the Plate Reader for the ADHP andmeasurements were taken.

ADHP—Excitation=550 nm; Emission=590 nm

Echip:

Chips used were 16-microelectode array composed of evaporated gold,ablated into the desired circuit. The structure of PB65-33 is:

Materials/SAM Solution Preparation

EAM: Ratio (1 mg/1 mL EtOH) PEG3-C11-SH (EAM:D1) PB65-33 MW: 336.53Ethanol 0.1:0.25 994 μL of 1.45 1212 μL of 2.97 mM stock 12.194 mLPB65_33: mM stock (11-mercaptoundecyl) MW 690.5 (EAM PB65-33):triethyleneglycol 0.5 mg aliquot (SENSOPATH) (1 mg/0.3 mg) Target 1xPBS, 100 mM High pH Washing Buffer: Incubation NaHCO₃ pH 10 Buffer:Testing Buffer: 1M LiClO₄ • 3H₂O MW: 160.44 Washing Buffer Nanopurewater Electrodes: External Quasi 10 chips and platinum wire

PEG3-C11-SH: (11-mercaptoundecyl)triethylenedlycol (SENSOPATH)

Day 1: SAM Deposition

-   1.1 For all chips, the following procedure was performed to deposit    the SAM-   1.2. Chips were rinsed again with ethanol and dried with argon-   1.3. Accessory parts (gasket, tubs) cleaned by washing machine,    rinsed with ethanol and air-dried. Bases were cleaned by hand.-   1.4. 200 μL of the deposition solution prepared above was added to    each chip.-   1.5. Chips were incubated overnight in ethanol incubation chamber

Day 2: Initial washing of chips

-   2.1 Following overnight incubation, chips removed from containers-   2.2 The chips were washed as follows:    -   2.2.1. 8× ethanol    -   2.2.2. 4× nanopure water

Target Incubation

4.1. Each chip was incubated as designated in table below. Buffer usedfor the samples was complete lysis buffer with modified Phosphataseinhibitor with zinc acetate and sodium azide. The samples were pHadjusted to pH 10 with 400 mM carbonate in (1:4 ratio)4.2. After target incubation of 10 min., the chips were washed 4× water,2×LiClO₄ and tested

Macro Chips - NADH (3 chips per conc) Solution Chip 1  1 mM Chip 2 300μM Chip 3 100 μM Chip 4  30 μM Chip 5  10 μM Chip 6  3 μM Chip 7  1 μMChip 8 300 nM Chip 9 100 nM  Chip 10  30 nM  Chip 11  0  Chip 12 UnknownCell lysate

Example 5 ATP Detection via Glycerol Kinase/Glycerol-3-Phosphate OxideSystem

ATP can be electrochemically detected by exploiting the ATP-dependentenzymatic reactions with a SAM-modified gold electrode by using theactivities of glycerol kinase (GK) and glycerol-3-phosphate oxidase(G3PO) if the co-substrate glycerol is present in the reaction mixture(see Murphy, L. J. et al. Anal. Chem. 1994, 66, 4345-4353; and Llaudet,E. et al., Anal. Chem. 2005, 77, 3267-3273).

First, GK catalyzes the formation of glycerol-3-phosphate from glyceroland ATP. Subsequently, G3PO oxidizes glycerol-3-phosphate to yieldglycerone phosphate and hydrogen peroxide. Thus, the amount of peroxideproduced is directly linked to the amount of ATP in the sample ifglycerol is in excess. An approach toward the electrochemical detectionof peroxide produced from this enzymatic reaction sequence is shown inFIG. 14.

The ATP sensor is based on a SAM-modified gold electrode that containsperoxide-reactive electroactive molecules (EAMs). The initial apparentformal potential of EAMs in the monolayer characterizes the sensor “off”state. Upon exposure to an ATP-containing sample matrix, hydrogenperoxide production triggers an irreversible elimination reaction in theimmobilized EAMs resulting in a signal “on” change in apparent formalpotential of ferrocene groups that is detected electrochemically. Thisenzyme-triggered redox altering chemical elimination (E-TRACE) reactionfor a representative class of ferrocene EAMs is shown in FIG. 11. (seee.g. U.S. patent application Ser. No. 12/853,204).

Purpose:

The purpose of this experiment was to demonstrate that ATP levelsdetected in cell lysates matched the same value on the standard curve inthe amplified and unamplified detection assays.

Materials and Methods:

Glycerol (2 mM), Glycerol kinase (GK, 1.33 U/mL), Glycerol 3-phosphateoxidase (G3PO, 1.33 U/mL), ATP (0 nM-1 mM)

Buffers Used:

0.5×HEPES, pH 7.4 (29 mM Maltoside)

1:200 Benzonase

1:2000 Protease inhibitor

1:100 Phosphatase Inhibitor cocktail (0.5×)

2.5 mM NaF

3 μM Sodium Orthovanadate

500 μM Sodium Pyrophosphate decahydrate

500 μM beta-Glycero-phosphate

100 μM Zn acetate

-   -   2.5 mM NaN₃ added to the phosphatase inhibitor cocktail as a        catalase inhibitor for H₂O₂

ATP: 0 nM-1 mM→E-TRACE Assay Only (in Triplicate)

ATP dilutions were made in nanopure water. This was used to make the ATPserial dilutions, and reconstitute GK and G3PO enzymes.

Desired Concentrations in Well/Tube

Solution Concentration ATP   0 nM-1 mM Glycerol   2 mM Glycerol kinase(1x) 1.33 U/mL Glycerol 3-phosphate 1.33 U/mL oxidase (1x) ADHP  160 μMHRP - 8x 0.16 U/mL

Buffer Types Concentration of components Buffer A - HEPES, pH 7.4 0.5xHEPES 25 mM, NaCl - 100 mM (Maltoside 29 mM) Inhibitors 1:200 Benzonaseadded Phosphatase inhibitor cocktail 0.5x (3 μM orthovanadate) - insolution (modified) (different prep) NaN₃ 2.5 mM Zn acetate 100 μMProtease inhibitor cocktail 1:2000 (Calbiochem 539134 VWR 80053-85

Assay volume will be 100 μL.

GK: 10 U/Aliquot

Reconstitute in 250 μL buffer-0.04 U/μL. Take 5 μL of 0.04 U/μL stockand add to each well—final assay concentration—1×. (For 100 μL assayvolume, add 3.57 μL of GK for each data point)

G3PO: 5 U/Aliquot

Reconstitute in 125 μL buffer=0.04 U/μL. Take 5 μL of 0.04 U/μL stockand add to each well—final assay concentration—1.33 U/mL (1×). (For 100μL assay volume, add 3.57 μL of G3PO for each data point)

Glycerol: 2 mM (MW—92.09 g/mol, density—1.261 g/cm³)

(2 mM)(150 μL)=(5 μL) (x)→x=60 mM needed

Take 22 μL of Glycerol stock and add to 4978 μL buffer to make a 5 mLtotal stock (60 mM Glycerol stock)

Take 3.57 μL and add to each well to make 100 μL total volume

Benzonase: 1:200 in 0.5×HEPES buffer, pH 7.4

Take 5 μL of Benzonase stock and add to 1 mL of 0.5×HEPES, pH 7.4 buffer

Phosphatase inhibitor cocktail (modified) 1:100—Weighed out each of thecomponents and added

1) 250 mM NaN₃ to 5 mL (50×) phosphatase inhibitor cocktailsolution—such that final concentration in 0.5× buffer will have 2.5 mMNaN₃.2) Also made a 1M stock of Zn acetate and added 1 μL of 1M stock to 100μL of 50× Phosphatase inhibitor cocktail aliquot—such that finalconcentration of Zn acetate in 0.5× lysis buffer=100 μM.

Add ( 1/100)*1 mL=10 μL of phosphatase inhibitor cocktail added to0.5×HEPES, lysis buffer

Protease inhibitor (1:2000 dilution)—in 0.5×HEPES, pH 7.4

( 1/2000)*1 mL=0.5 μL Protease inhibitor added to 1 mL 0.5×HEPES, lysisbuffer

Assay conditions: (GK—1×, G3PO—1×)—HEPES 0.5× (0.5×Phosphataseinhibitor)

100 μL of each sample was incubated for 2.5 hrs. The followingcomponents were added to the tube for 2.5 hrs incubation before puttingthem on chips for target testing.

Upon incubation in 0.5×HEPES, pH 7.4 for 2.5 hrs, adjust pH to 10 byadding 33.3 μL 400 mM Na₂CO₃ (100 mM in assay)

Total 133.3 μL sample (per data point)—25% sample dilution occurs in theprocess of adjusting to pH 10

Add 30 μL sample to each chip all at once and incubate (for 10 min.) andwash them before reading

0 nM-1 mM Components added ATP Buffer   90 μL GK 3.57 μL G3PO 3.57 μLGlycerol (2 mM) 3.57 μL ATP   10 μL

E-Chip Experiment:

Chips used were 16-microelectode array composed of evaporated gold,ablated into the desired circuit. Structure of PB65-33:

Run e-chip on full ATP curve (0 nM-1 mM)

Average Peak current ratio and Area ratio were analyzed for allconcentrations.

Chips were washed 8× ethanol, 4× with water and incubated with targetsolution of 10 min. right away

Upon 10 min. incubation, chips were washed 4× water and 2×LiClO₄ andtested with 1 M LiClO₄ testing solution.

Results are shown in FIG. 16. From this experiment it was demonstratedthat the lysate concentration of ATP is roughly 1 μM.

1. A method for detecting a target analyte in a test sample, said methodcomprising: (a) providing a solid support comprising an electrodecomprising: (i) a self-assembled monolayer (SAM), (ii) a covalentlyattached electroactive active moiety (EAM) comprising a transition metalcomplex comprising a self-immolative moiety and a peroxide sensitivemoiety (PSM), wherein said EAM has a first E⁰, and (b) contacting saidtarget analyte and said solid support under conditions wherein saidtarget analyte reacts with a peroxide generating enzyme to generateperoxide and said self-immolative moiety is removed such that said EAMhas a second E⁰ test sample; and (c) detecting said second E⁰ as anindication of the presence of said target analyte.
 2. A method fordetecting a target analyte in a test sample, said method comprising: (a)providing a solid support comprising an electrode comprising: (i) aself-assembled monolayer (SAM), (ii) a covalently attached electroactiveactive moiety (EAM) comprising a transition metal complex comprising aself-immolative moiety and a peroxide sensitive moiety (PSM), whereinsaid EAM has a first E⁰, and (b) contacting said target analyte and saidsolid support in the presence of an intermediary enzyme and optionallyan additional substrate for said enzyme, under conditions wherein ifsaid analyte is present, the target analyte and said optional additionalsubstrate reacts with the intermediary enzyme to form a first complex;(c) contacting said first complex with said peroxide generating enzymeunder conditions wherein if said first complex is formed, said firstcomplex reacts with said peroxide generating enzyme to generate peroxideand said self-immolative moiety is removed such that said EAM has asecond E⁰ test sample; and (d) detecting said second E⁰ as an indicationof the presence of said target analyte.
 3. The method according to claim1, wherein said peroxide generating enzyme is immobilized or physisorbedonto the solid support, the electrode, or SAM.
 4. The method accordingto claim 1, wherein said intermediary enzyme is immobilized orphysisorbed onto the solid support, the electrode, or SAM.
 5. The methodaccording to claim 2, wherein steps (b) and (c) are carried outseparately.
 6. The method according to claim 2, wherein steps (b) and(c) are carried out simultaneously.
 7. The method according to claim 2,wherein said target analyte is ATP, said intermediary enzyme is glycerolkinase and said substrate of said intermediary enzyme is glycerol, andsaid peroxide generating moiety is glycerol-3-oxidase.
 8. The methodaccording to claim 1, wherein said target analyte is NADH and saidperoxide generating moiety is NADH oxidase (NAOX).
 9. The methodaccording to claim 1, wherein said solid support comprises an array ofelectrodes.
 10. The method according to claim 1, wherein said transitionmetal is selected from the group consisting of iron, ruthenium andosmium.
 11. The method according to claim 1, wherein said EAM is aferrocene.
 12. The method according to claim 1, wherein said detectingis done amperommetrically at each first and second E⁰.
 13. A compositioncomprising a solid support comprising: (a) an electrode comprising: (i)a self-assembled monolayer (SAM); (ii) a covalently attachedelectroactive active moiety (EAM) comprising a transition metal complexcomprising a self-immolative moiety and a peroxide sensitive moiety(PSM), wherein said EAM has a first E⁰ when said self-immolative moietyis covalently attached to said EAM and a second E⁰ when saidself-immolative moiety is absent; (iii) a capture binding ligandcomprising a substrate of an ATP-dependent intermediary enzyme; and (d)a soluble capture ligand comprising a peroxide generating moiety.
 14. Acomposition comprising a solid support comprising: (a) an electrodecomprising: (i) a self-assembled monolayer (SAM); (ii) a covalentlyattached electroactive active moiety (EAM) comprising a transition metalcomplex comprising a self-immolative moiety and a peroxide sensitivemoiety (PSM), wherein said EAM has a first E⁰ when said self-immolativemoiety is covalently attached to said EAM and a second E⁰ when saidself-immolative moiety is absent; and (iii) a peroxide generatingenzyme.